Poly(beta-amino esters) and uses thereof

ABSTRACT

Provided herein are branched poly(beta-amino esters) (PBAE) useful as vehicles for the delivery of therapeutic agents, such as nucleic acids. The disclosed polymers form stable compositions, and are suitable for the delivery of therapeutic agents via nebulization. Compositions of the disclosed polymers are capable of delivering therapeutic agents such as mRNA to lung epithelial cells.

RELATED APPLICATIONS

The present application is a division of U.S. application Ser. No.16/170,318, filed Oct. 25, 2018, which claims priority under 35 U.S.C. §119(e) to U.S. provisional application U.S. Ser. No. 62/578,139, filedOct. 27, 2017, each of which is incorporated herein by reference.

BACKGROUND

Nucleic acid-based therapeutics hold the potential to treat any diseasewith a protein target. DNA has been used for the majority of genetherapy clinical trials. The use of mRNA instead of DNA would negate therisk of insertional mutagenesis and also confer the ability to transfectnon-dividing cells which would be an advantage, particularly inrespiratory epithelium which is slowly dividing or terminallydifferentiated. For example, the ability of in vitro transcribed(IVT)-mRNA to restore surfactant protein B expression has beendemonstrated in the lung, albeit using invasive intra-tracheal delivery.The development of effective delivery systems for IVT-mRNA, and ways toaddress the immunogenicity and instability of IVT-mRNA, remain acritical hurdle for clinical adoption.

Branched polyethylenimine (bPEI) are known to be efficient deliveryvectors for nebulized gene delivery. However, toxicity concerns relatedto bPEI remain, due to accumulation of the relatively large,non-degradable polymer. Lower molecular weight PEIs tend to have lowertoxicity. However, DNA transfection efficiency is generally diminishedwith lower molecular weight PEIs, and those with molecular weights belowapproximately 1.8 kDa are ineffective. The immunogenicity andinstability of IVT-mRNA also is an obstacle to its therapeuticpotential. As such, there remains a need for effective, non-invasivevehicles for the delivery of therapeutic agents, such as nucleic acids.In particular, there remains a need for delivery vehicles that formstable compositions, suitable for the delivery of therapeutic agents,such as mRNA, via nebulization.

SUMMARY

The present disclosure relates to poly(beta amino esters) (PBAE)polymers useful for the non-viral delivery of agents (e.g., nucleicacids) to cells. The PBAE of Formula (I) and Formula (II), saltsthereof, and embodiments described herein, are collectively referred toas “polymers of the invention.” The polymers of Formula (I) are branchedor hyperbranched. The polymers of Formula (II) are linear. The degree ofbranching can be used to optimize properties such as solubility,viscosity, and efficacy as a transfection reagent. Polymers of theinvention can be used to prepare stable formulations (e.g., particles)for nebulization or aerosol delivery.

In one aspect, provided herein are poly(beta-amino esters) (PBAE) ofFormula (I), comprising: a bis(propionyl) diradical of Formula (A), adiradical of Formula (B), a triradical of Formula (C), a radical ofFormula (D), and optionally, a diradical of Formula (E):

In another aspect, provided herein are compositions comprising a polymerof Formula (I) and an agent. In certain embodiments, the agent is aprotein, peptide, polynucleotide, or small molecule.

In another aspect, provided herein are poly(beta-amino esters) (PBAE) ofFormula (II), comprising: a bis(propionyl) diradical of Formula (A), adiradical of Formula (B), a radical of Formula (D), and optionally, adiradical of Formula (E).

Provided in other aspects are methods of delivering a polynucleotide toa cell by contacting the cell with a composition comprising a polymer ofFormula (I) or Formula (II), and methods of treating a disease ordisorder in a subject in need of such treatment by administering to thesubject a pharmaceutical composition comprising a polymer of Formula (I)or Formula (II).

In another aspect, provided herein a method of preparing a polymer ofFormula (I), comprising (i) combining a bis(acrylate) compound ofFormula (A′) with a compound of Formula (B′), a compound of Formula(C′), and optionally a compound of Formula (E′); and (ii) combining theproduct of (i) with a compound of Formula (D′):

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. FIG. 1A shows a subset of diacrylates, primary/bis-aminesand end-cap amine monomers used to synthesize linear PBAEs. FIG. 1Bshows a representative synthesis scheme. FIG. 1C shows transfectionresults for the following: A549 lung epithelial cells transfected with50 ng of mRNA encoding for firefly luciferase and bioluminescence,assessed after 24 hours, normalized to cell viability (n=4, +S.D.). Thehighest luminescence was observed in cells that were transfected with aPBAE composed of monomers DD90-118. The effect of molecular weight, endcap amine, and addition of alkylamine C12 in vivo can be found in FIGS.11 and 12B.

FIGS. 2A-2B. FIG. 2A shows the synthesis of hyperbranched DD90-118 PBAEs(hDD90-118) via the addition of a tri-functional amine (BB′2) to DD90followed by end-capping with monomer 118. FIG. 2B shows that thetransfection efficiency of hyperbranched PBAEs in A549 lung epithelialcell line was assessed by measuring bioluminescence 24 hours afterdelivery of mRNA encoding for firefly luciferase normalized to cellviability (n=3, +S.D.). The hPBAEs of FIG. 2B were prepared according toExample 2.

FIGS. 3A-3C. FIG. 3A shows particle stability at various pHs.Hyperbranched PBAE hDD90-118 (0.3) and linear DD90-118 both begin toaggregate as pH increases. Nanoparticles composed of the linear PBAEbegin to increase in size at pH 7.5 and aggregates above pH 8, whereasnanoparticles of the hyperbranched PBAE are stable with respect toparticle diameter until pH 8.5 and aggregate above pH 9. FIG. 3B showsthat this loss of particle stability in FIG. 3A correlates to areduction in zeta potential below +30 mV with increasing pH. FIG. 3C isdynamic light scattering of DD90-118 or C32-118 based polymers complexedwith mRNA (50 to 1) at two concentrations; ‘high’ 0.5 mg/mL mRNA (dashedlines) and low′ 0.003 mg/mL mRNA (solid lines). At pH 7.4, linear PBAEsformulated with mRNA at 0.5 mg/mL (grey dashed line) become unstable andaggregate into large particles whereas hyperbranched analogues (blackdashed line) remain stable nanoparticles below 200 nm. Both linear andbranched PBAEs at a mRNA concentration of 0.003 mg/mL remain stable(grey and black solid lines, respectively).

FIGS. 4A-4H. FIG. 4A depicts a schematic of the vibrating mesh nebulizerand whole body chamber used for aerosol delivery of mRNA encodingfirefly luciferase. As shown in FIG. 4B, radiance was measured andprotein quantified in the lungs of C57BL/6 mice 24 hours afternebulization of 0.5 mg/mL of mRNA n=3-6. FIG. 4C shows that uniformdistribution of radiance was observed across 5 lobes of the lung treatedwith hDD90-118. FIG. 4D shows that bioluminescence was localized to thelung with no radiance detected in other major organs. As shown in FIG.4E, hDD90-118 nanoparticles can be repeatedly administered vianebulization (green arrows) and facilitates repeated production ofluciferase protein in the lung. There was no significant weight loss orinflammation after repeated inhaled delivery of hDD90-118 nanoparticles(FIG. 4F and FIG. 4G) in any of the treatment groups. Flow cytometryanalysis of the lung found that the epithelial cell population was themajority cell sub-type transfected by hDD90-118 nanoparticles afternebulization of mRNA encoding for Cre-recombinase to Ai14 tdTomatoreporter mice (+S.D, n=3), FIG. 4H.

FIGS. 5A-5E. FIG. 5A shows intravenous delivery of mRNA (0.5 mg/kg)encoding for firefly luciferase via tail vein injection leads tobioluminence in the lungs and spleen. FIG. 5B shows that highestradiance is observed in the lungs of mice transfected with hDD90-118with DB 0.3 (hPBAE) nanoparticles (*p<0.05), (medians±IQR, n=4),toxicity as assessed by weight loss is significantly lower in hPBAEcompared to the linear analogue shown in FIG. 5C. Statistical analysisusing one-way Anova with Tukeys test (+S.D, n=4). FIG. 5D and FIG. 5Eshows flow cytometry of lung cells after intravenous injection, lungendothelial cells are the majority sub-type transfected by hDD90-118(hPBAE) and branched polyethenylenimine 25 kDa (bPEI). (n=3, +S.D).

FIGS. 6A-6C. FIG. 6A shows the relative amount of acrylate (DD) andbackbone amine (90) as determined using peaks in ¹H NMR spectra at 7.09ppm ‘a’ and 3.49 ppm ‘c’ respectively. Branching amine was determinedusing peak at 2.17 ppm (D) and terminal end-cap amine (118) determinedusing peak at 0.89 ppm (E). FIG. 6B shows the molar ratio of thesemonomers in PBAE as determined by ¹H NMR; as the degree of branchingincreases, the ratio of end cap amine to acrylate also increases. FIG.6C shows the degree of branching (DB), which was calculated according tothe equation DB=(D+T)/(D+T+L)* where D is the number of dendritic units(e.g., branching amine of Formula (C)), T is terminal units (118), and Lis linear units (DD and 90). Theoretical values calculated from feedratios during reaction are compared to values determined experimentallyby ¹H NMR and show good agreement. Hyperbranched DD90-118 with varyingdegrees of branching were synthesized by changing feed ratio of monomer90 to monomer BB2′. (*Hawker & Fretchet 1991 J. Am. Chem. Soc. 113(12)).

FIGS. 7A-7B. FIG. 7A shows triple detection gel permeationchromatography (GPC), example chromatograms for hDD90-118 (0.2). FIG. 7Bshows molecular weight (Mw) and intrinsic viscosity determined by GPC.As branching of the polymer increases, viscosity is reduced.

FIG. 8 shows that gel electrophoresis was used to assess ability ofnanoparticle complex to retard mobility. Linear DD90-118 (Lin) andhyperbranched hDD90-118 with degree of branching 0.3, 0.2, and 0.1 arecomplexed with mRNA at three mass ratios of polymer to nucleic acid 2.5to 1, 5 to 1, and 7.5 to 1. None of the polymers could retard mRNA at aratio of 2.5 to 1. At a ratio of 5 to 1, hDD90-188 with a DB of 0.3could retard mRNA greater than the other PBAEs. All hyperbranchedpolymers could retard mobility at 7.5 to 1 but not linear.

FIGS. 9A-9B depict the aqueous solubility at pH 5.2. FIG. 9A shows acalibration curve, which demonstrates a linear relationship betweenpolymer concentration and absorbance at 250 nm. In FIG. 9B, serialdilutions of a supersaturated solution were filtered and absorbancemeasured at 250 nm. The average original concentration of dilutedsamples was calculated and found to be greater for hyperbranched PBAEcompared to linear (n=3, +S.D.).

FIGS. 10A-10C depict stability and efficacy after storage of lyophilizedsamples at −80° C. (n=3, ±S.D). Zeta potential (FIG. 10A), diameter(FIG. 10B), and radiance (FIG. 10C) were measured.

FIG. 11 shows the average radiance measured in the mice afternebulization. Linear PBAEs were nebulized 0.25 mg/mL, 2 mL total (0.5mg) mRNA to three mice.

FIG. 12 shows the average radiance measured in three mice after theaddition of PEG-lipid and a lower dose of mRNA 0.15 mg.

DETAILED DESCRIPTION

The present disclosure relates to inventive poly(beta amino esters)(PBAE), which useful for the non-viral delivery of agents (e.g., nucleicacids) to cells. In certain embodiments, polymers of the invention areuseful for the delivery of mRNA to both lung endothelium and epitheliumvia nebulization, dry powder inhalation or systemic administration andtherefore are clinically relevant to the treatment of disorders of thelung epithelium, including enzyme deficiencies and cystic fibrosis.

Polymers of Formula (I)

In one aspect, provided herein is a poly(beta-amino ester) (PBAE)polymer of Formula (I) comprising:

a. a bis(propionyl) diradical of Formula (A):

b. a diradical of Formula (B):

c. a triradical of Formula (C):

d. a radical of Formula (D):

and optionally

e. a diradical of Formula (E):

wherein:

R is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedarylene, optionally substituted heteroarylene, or a combination thereof;

R_(1a), R_(1b), R₂, R₃, R_(4a), and R_(4b) are each independentlyhydrogen, halogen, hydroxyl, alkoxyl, cyano, optionally substitutedaliphatic, or optionally substituted heteroaliphatic;

R₅ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedaryl, or optionally substituted heteroaryl;

R₆ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedaryl, or optionally substituted heteroaryl;

R₇ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted optionallysubstituted arylene, or optionally substituted heteroarylene;

R₈ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted optionallysubstituted arylene, or optionally substituted heteroarylene; and

R₉ is optionally substituted aliphatic;

wherein:

each Formula (A) has two points of attachment to radicals selected fromFormulae (B), (C), (D), and optionally (E);

each Formula (B) has two points of attachment to radicals of Formula(A);

each Formula (C) has three points of attachment to radicals of Formula(A);

each Formula (D) has one point of attachment to a radical of Formula(A); and

each Formula (E), if present, has two points of attachment to radicalsof Formula (A);

and wherein Formulae (B), (C), (D), and (E), if present, are differentfrom one another.

The polymer of Formula (I) comprises radicals of Formulae (A), (B), (C),(D) and optionally (E) in various orders, arrangements, and molarpercentages. By varying the molar percentages of one or more of thecomponent radicals, it is possible to control the degree of branching,relative abundance of primary amines (e.g., primary amine termini) andthereby to control properties such as aqueous solubility andtransfection efficiency.

In certain embodiments, the polymer of Formula (I) comprises a radical(i.e., a mono-radical or poly-radical) selected from the radicals ofTable 1, and combinations thereof:

TABLE 1 (1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10) 

(11) 

(12) 

(13) 

(14) 

(15) 

(16) 

(17) 

(18) 

For example, radical (18) consists of radicals of Formulae (D), (A), and(C) in the indicated arrangement. Radical (18) may also be representedby the following formulae:

and

In certain embodiments, the polymer of Formula (I) comprises radicals ofFormulae (A) and (B) in a molar ratio of about 1:0.5 to about 1:1. Incertain embodiments, the polymer of Formula (I) comprises radicals ofFormulae (A) and (C) in a molar ratio of about 1:0 to about 1:0.2. Incertain embodiments, the polymer of Formula (I) comprises radicals ofFormulae (A) and (D) in a molar ratio of about 1:0.5 to about 1:0.4. Incertain embodiments, the polymer of Formula (I) comprises radicals ofFormulae (A), (B), (C) and (D) in a molar ratio of about 1:0.5:0.2:0.39;1:0.67:0.13:0.27; 1:0.8:0.08:0.16; or 1:0.94:0:0.07. In certainembodiments, the polymer of Formula (I) comprises radicals of Formulae(A), (B), (C), (D) and (E) in a molar ratio of about1:0.35:0.2:0.39:0.15; 1:0.47:0.13:0.27:0.2; 1:0.56:0.08:0.16:0.24; or1:0.66:0:0.07:0.28.

In certain embodiments, the polymer of Formula (II) comprises a linearradical selected from Table 1.

Polymers of Formula (II)

In another aspect, provided herein is a poly(beta-amino ester) (PBAE)polymer of Formula (II) comprising:

a. a bis(propionyl) diradical of Formula (A):

b. a diradical of Formula (B):

c. a radical of Formula (D):

and optionally

d. a diradical of Formula (E):

wherein:

R is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedarylene, optionally substituted heteroarylene, or a combination thereof;

R_(1a), R_(1b), R₂, R₃, R_(4a), and R_(4b) are each independentlyhydrogen, halogen, hydroxyl, alkoxyl, cyano, optionally substitutedaliphatic, or optionally substituted heteroaliphatic;

R₅ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedaryl, or optionally substituted heteroaryl;

R₈ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted optionallysubstituted arylene, or optionally substituted heteroarylene; and

R₉ is optionally substituted aliphatic;

wherein:

each Formula (A) has two points of attachment to radicals selected fromFormulae (B), (D), and optionally (E);

each Formula (B) has two points of attachment to radicals of Formula(A);

each Formula (D) has one point of attachment to a radical of Formula(A); and

each Formula (E), if present, has two points of attachment to radicalsof Formula (A);

and wherein Formulae (B), (D), and (E), if present, are different fromone another.

In certain embodiments, Formulae (A), (B), (D) and (E) are as definedherein.

In certain embodiments, the polymer of Formula (II) comprises radicalsof Formulae (A) and (B) in a molar ratio of about 1:0.5 to about 1:1. Incertain embodiments, the polymer of Formula (II) comprises radicals ofFormulae (A) and (D) in a molar ratio of about 1:0.5 to about 1:0.4. Incertain embodiments, the polymer of Formula (II) comprises radicals ofFormulae (A), (B), and (D) in a molar ratio of about 1:0.5:0.625 to1:1:0.125. In certain embodiments, the polymer of Formula (II) comprisesradicals of Formulae (A), (B), (D) and (E) in a molar ratio of about1:0.35:0.625:0.15: to 1:0.7:0.125:0.3.

Molecular Weight

Molecular weight is influenced by the molar ratio of diacrylate monomercorresponding to the diradical of Formula (A) to the amine monomerscorresponding to Formulae (B) and optionally (E). A higher molar ratioof diacrylate monomer amine monomer will lead to lower molecular weight.A diacrylate:amine molar ratio of about 1 will lead to greater molecularweight.

In certain embodiments, the polymers of the invention have a molecularweight in the range of 1-100 kDa, 5-50 kDa, 10-40 kDa, or 15-30 kDa. Incertain embodiments, the polymers of the invention have a molecularweight in the range of 15-24 kDa. In certain particular embodiments, thepolymers of the invention have a molecular weight of about 15 kDa, about16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21kDa, about 22 kDa, about 23 kDa, or about 24 kDa.

Branching

As used herein, the term “branched” refers to polymers containingbranches that are composed of the same units that make up the linearportion of the main chain. As used herein, the term “hyperbranched”refers to polymers containing branches that are composed of the unitsthat make up the linear portion of the main chain as well as furtherbranch points (e.g., radicals of Formula (C), also referred to as“dendritic units”). Hyperbranched dendritic polymers contain randomlydistributed dendritic units and offer a large chemical space forinvestigation as they can be synthesized with a wide range of monomersusing one-pot reaction conditions. Linear segments can be combined withhyperbranched segments to alter the degree of branching (DB), therebyaltering properties such as solubility, viscosity, and efficacy as atransfection reagent.

The terms “Degree of branching” and “DB” can be defined as the ratio ofdendritic units (radicals of Formula (C)) to linear units (radicals ofFormulae (A), (B), (D), and optionally (E)). DB can be calculated usingthe equation: DB=(D+T)/(D+T+L), where D is number of dendritic units, Tis the number of terminal units (radical of Formula (D)) and L is numberof linear units (radicals of Formulae (A), (B), and optionally (E)).(See also, Hawker & Fretchet 1991 J. Am. Chem. Soc. 113(12)). DB can becontrolled as a function of the stoichiometry of Formula (B) to Formula(C). For example, higher DB is obtained by using a molar excess of themonomer corresponding to Formula (B), relative to the monomercorresponding to Formula (C). Linear polymers (DB=0) are obtained byomitting the monomer corresponding to Formula (C).

DB also correlates directly with an increase in terminal primary aminegroups. Increased density of primary amines in the PBAE may influencepolymer efficacy as a transfection reagent at various stages during theformulation and transfection process, for example, during nanoparticleformulation when the cationic polymer protects nucleic acid cargothrough electrostatic condensation to prevent degradation by nucleases.

During endocytosis of the nanoparticle, increased amine density alsoincreases the capacity of the polymer to become protonated, contributingto the “proton sponge” mechanism that has been proposed to triggerendosomal disruption, and subsequent escape into the cytoplasm, criticalfor translation of mRNA.

In certain embodiments, the polymers of the invention are linear. Incertain embodiments, the polymers of the invention are branched orhyperbranched. DB influences properties such as intrinsic viscosity,solubility, and transfection efficacy. In certain embodiments, thepolymer of Formula (I) has a degree of branching (DB) in the range of0.0-1.0. In certain embodiments, the polymer of Formula (I) has a degreeof branching (DB) in the range of 0.0-0.5. In certain particularembodiments, the DB of a polymer of Formula (I) is 0.0, about 0.1, about0.2, about 0.3, about 0.4, about 0.5, about 0.6, or about 0.7.

In certain embodiments, the polymers of the invention are soluble in anaqueous solution. The aqueous solvent may comprise of 50-100% water byvolume. In a particular embodiment, the aqueous solvent comprises atleast 90%, at least 92%, at least 94%, at least 96%, at least 98% or atleast 99% water by volume. In certain embodiments, the pH of the aqueoussolvent is in the range of about 5 to about 7.5 (e.g., about 5.2 toabout 7.4). In certain embodiments, the temperature of the aqueoussolution is in the range of about 25° C. to about 37° C. An aqueoussolution may further comprise an organic solvent such asdimethylsulfoxide, dimethylformamide, acetic acid, or an alcohol (e.g.,methanol, ethanol or isopropanol).

In certain embodiments, the polymers of the invention have a solubilityin aqueous solution of at least about 30, at least about 10, at leastabout 5, at least about 2, or at least about 1 mg/mL. In certainparticular embodiments, the polymer has a solubility in an aqueoussolution of at least about 1.6 mg/mL at about pH 7.4.

In certain embodiments, the polymers of the invention are biodegradableor biocompatible. As used herein, “biodegradable” polymers are thosethat, when introduced into cells, are broken down by the cellularmachinery or by hydrolysis into components that the cells can eitherreuse or dispose of without significant toxic effect on the cells (i.e.,fewer than about 20% of the cells are killed when the components areadded to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain embodiments,the chemical reactions relied upon to break down the biodegradablepolymers are uncatalyzed. Biodegradability is a particular advantage ofthese PBAE delivery vectors, particularly for repeat administrationwhere non-degradable vectors like PEI may accumulate or be difficult forthe body to metabolize. The term “biocompatible,” as used herein isintended to describe compounds that are not toxic to cells. Polymers are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and their administration in vivo does notinduce inflammation or other such adverse effects.

Formula (A)

Formula (A) is a bis(propionyl) diradical, having two points ofattachment to radicals selected from Formulae (B), (C), (D), andoptionally (E).

In certain embodiments, Formula (A) is:

In certain embodiments, Formula (A) is of Formula (A1), (A2), (A3) or(A4):

In certain embodiments, R is optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substitutedcarbocyclylene, optionally substituted heterocyclylene, optionallysubstituted arylene, optionally substituted heteroarylene, or acombination thereof.

In certain embodiments, R is substituted aliphatic, unsubstitutedaliphatic, substituted heteroaliphatic, unsubstituted heteroaliphatic,substituted arylene, unsubstituted arylene, substituted heteroarylene,unsubstituted heteroarylene or a combination thereof.

In certain embodiments, R is selected from carbon chains of 1 to 30carbon atoms, heteroatom-containing carbon chains of 1 to 30 atoms, andcarbon chains and heteroatom-containing carbon chains with at least onesubstituent selected from the group consisting of branched andunbranched alkyl, branched and unbranched alkenyl, branched andunbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino,ureido, cycloalkyl, heterocyclyl, aryl, heteroaryl, halogen, hydroxyl,alkoxy, cyano, amido, carbamoyl, carbonyl, carbonyldioxyl,alkylthioether, and thiol groups.

In certain embodiments, R_(1a), R_(1b), R₂, R₃, R_(4a), and R_(4b) areeach independently hydrogen, halogen, hydroxyl, alkoxyl, cyano,optionally substituted aliphatic, or optionally substitutedheteroaliphatic.

In certain embodiments, R_(1a), R_(1b), R₂, R₃, R_(4a), and R_(4b) areindependently selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkoxyl, carbamoyl, carbonyldioxyl,amido, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino,trialkylamino, cyano, and ureido, each of which may be substituted, aspermitted by valency, with at least one substituent selected from thegroup consisting of alkyl, alkenyl, alkynyl, amino, alkylamino,dialkylamino, trialkylamino, aryl, ureido, cycloalkyl, heterocyclyl,aryl, heteroaryl, halogen, hydroxyl, alkoxy, cyano, amido, carbamoyl,carbonyl, carbonyldioxyl, alkylthioether, and thiol groups.

In certain embodiments, R^(a), R^(b), R^(c), R^(d), and Re areindependently selected from optionally substituted C₁₋₆ alkyl,optionally substituted C₃₋₇ cycloalkyl, optionally substituted C₂₋₆alkenyl, optionally substituted C₁₋₆ heteroalkyl, optionally substitutedC₂₋₆ heterocyclyl, optionally substituted C₆₋₁₀ aryl, or optionallysubstituted C₂₋₆ heteroaryl.

In certain embodiments, n is 2-10. In certain particular embodiments, nis 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In certain embodiments of Formulae (A), (A1), and (A3), R_(1a) andR_(4a) each are hydrogen. In certain embodiments, R_(1a), R_(1b), R_(4a)and R_(4b) each are hydrogen. In certain embodiments of Formulae (A),(A1), and (A3), R₂ and R₃ each are hydrogen. In certain embodiments,R_(1a), R_(1b), R₂, R₃, R_(4a) and R_(4b) are all hydrogen.

In certain particular embodiments, Formula (A) has the structure:

In certain particular embodiments, Formula (A) has the structure:

In certain embodiments, the polymers of the invention (e.g., a polymerof Formula (I) or (II)) comprise about 30-60 mol % Formula (A). Incertain embodiments, the polymers of the invention comprise about 20-50mol % Formula (A). In certain embodiments, the polymers of the inventioncomprise about 44-48 mol % Formula (A). In certain particularembodiments, the polymers of the invention comprise 44, 45, 46, 47 or 48mol % Formula (A).

In certain embodiments, all radicals of Formula (A) are the same. Inother embodiments, there are two or more (e.g., 2, 3 or 4) differentradicals of Formula (A).

Formula (B)

Formula (B) is a diradical having two points of attachment to radicalsof Formula (A). Typically Formula (B) joins to two different Formula (A)moieties.

In certain embodiments, Formula (B) is:

In certain embodiments, Formula (B) is of Formula (B1), (B2) or (B3):

In certain embodiments, R₅ is optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedoptionally substituted aryl, or optionally substituted heteroaryl.

In certain embodiments, R₅ is alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkoxy, carbamoyl, carbonyldioxyl, amido, thiohydroxyl,alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano,ureido, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, each ofwhich may be substituted with at least one substituent selected from thegroup consisting of alkyl, alkenyl, alkynyl, amino, alkylamino,dialkylamino, trialkylamino, aryl, ureido, cycloalkyl, heterocyclyl,aryl, heteroaryl, halogen, hydroxyl, alkoxy, cyano, amido, carbamoyl,carbonyl, carbonyldioxyl, alkylthioether, and thiol groups.

In certain embodiments, R_(5a) is optionally substituted alkylene, oroptionally substituted heteroalkylene.

In certain embodiments, R_(5b) is absent, or is optionally substitutedC₃₋₇ cycloalkyl, optionally substituted C₁₋₆ heteroalkyl, optionallysubstituted C₂₋₆ heterocyclyl, optionally substituted C₆₋₁₀ aryl, oroptionally substituted C₂₋₆ heteroaryl.

In certain embodiments, R_(5c) is optionally substituted C₃₋₇cycloalkyl, or optionally substituted C₂₋₆ heterocyclyl.

In certain embodiments, Formula (B) is selected from the following:

In certain particular embodiments, Formula (B) has the structure:

In certain particular embodiments, Formula (B) has the structure:

In certain embodiments, the polymers of the invention (e.g., a polymerof Formula (I) or (II)) comprise about 10-60 mol % Formula (B). Incertain embodiments, the polymers of the invention comprise about 20-50mol % Formula (B). In certain embodiments, the polymers of the inventioncomprise about 24-42 mol % Formula (B). In certain particularembodiments, the polymers of the invention comprise 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 mol % Formula(B).

In certain embodiments, all radicals of Formula (B) are the same. Inother embodiments, there are two or more (e.g., 2, 3 or 4) differentradicals of Formula (B).

Formula (C)

Formula (C) is a triradical having three points of attachment toradicals of Formula (A). Triradicals of Formula (C) are branch points inPBAEs of the invention. In certain embodiments, the polymers of theinvention do not include a branch point of Formula (C).

In certain embodiments, Formula (C) is:

In certain embodiments, Formula (C) is of Formula (C1):

In certain embodiments, R₆ is optionally substituted C₁₋₆ alkyl.

In certain embodiments, R₇ is optionally substituted C₁₋₆ alkyl,optionally substituted C₃₋₇ cycloalkyl, or optionally substitutedC₁₋₆heteroalkyl.

In certain embodiments, Formula (C) is selected from:

In certain particular embodiments, Formula (C) has the structure:

In certain embodiments, the polymer of Formula (I) comprises about 0-50,about 0-40, about 0-30, about 0-20, or about 0-10 mol % Formula (C). Incertain embodiments, the polymer of Formula (I) comprises about 0-10 mol% Formula (C). In certain particular embodiments, the polymer comprises0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol % Formula (C).

Formula (D)

Formula (D) is a radical having one point of attachment to a radical ofFormula (A). Radicals of Formula (D) occur at the termini of PBAEpolymers of the invention.

In certain embodiments, Formula (D) is:

In certain embodiments, R₈ is optionally substituted aliphatic,optionally substituted heteroaliphatic, optionally substitutedcarbocyclylene, optionally substituted heterocyclylene, optionallysubstituted arylene, or optionally substituted heteroarylene. In certainembodiments, R₈ is optionally substituted aliphatic or optionallysubstituted heteroaliphatic. In certain embodiments, R₈ is optionallysubstituted C₁₋₆ alkylene, or optionally substituted C₁₋₆heteroalkylene.

In certain embodiments, Formula (D) is of Formula (D1):

wherein n, m, and p are each independently an integer between 0 and 20,inclusive; and V is —O—, —S—, —NH—, —NR_(V)—, or C(R_(V))₂, whereinR_(V) is hydrogen, hydroxyl, C₁₋₆aliphatic, C₁₋₆heteroaliphatic,C₁₋₆alkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, aryl, heteroaryl,thiol, akylthioxy, or acyl. In certain embodiments, n, m, p and q areeach independently an integer between 1 and 15, inclusive. In yet otherembodiments, n, m, p and q are each independently an integer between 1and 12, inclusive. In other embodiments, n, m, p and q are eachindependently an integer between 1 and 10, inclusive. In otherembodiments, n, m, p and q are each independently an integer between 0and 6, inclusive. In still other embodiments n, m, p and q are eachindependently an integer between 0 and 3, inclusive. In certainembodiments, n, m, p and q are each independently 0, 1, 2, 3, 4, 5, or6. In certain embodiments, the terminal amino group of Formula (D1) isalkylated (e.g., C₁-C₁₂ alkyl), acylated (e.g., acetyl), or otherwisemodified.

In certain embodiments, Formula (D) is of Formula (D2):

wherein m is an integer between 0 and 20, inclusive. In certainembodiments, m is an integer between 1 and 15, inclusive. In yet otherembodiments, m is an integer between 1 and 12, inclusive. In otherembodiments, m is an integer between 1 and 10, inclusive. In otherembodiments, m is an integer between 0 and 6, inclusive. In still otherembodiments, m is an integer between 0 and 3, inclusive. In certainembodiments, m is 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, theterminal amino group of Formula (D2) is protected, alkylated (e.g.,C₁-C₁₂ alkyl), acylated (e.g., acetyl), or otherwise modified.

In certain embodiments, Formula (D) is of Formula (D3):

wherein m is an integer between 1 and 20, inclusive. In certainembodiments, m is an integer between 2 and 15, inclusive. In yet otherembodiments, m is an integer between 2 and 12, inclusive. In otherembodiments, m is an integer between 2 and 10, inclusive. In otherembodiments, m is an integer between 2 and 6, inclusive. In still otherembodiments, m is 2 or 3.

In certain embodiments, Formula (D) is of Formula (D4):

wherein R_(8a) is optionally substituted C₁₋₆ alkyl, or optionallysubstituted C₁₋₆ heteroalkyl. R_(8a) is covalently attached to a carbonatom of the alkyl chain.

In certain embodiments, Formula (D) is selected from:

In certain particular embodiments, Formula (D) has the structure:

In certain embodiments, the polymer comprises about 1-25 mol % Formula(D). In certain embodiments, the polymer comprises about 5-20 mol %Formula (D). In certain embodiments, the polymer comprises about 8-19mol % Formula (D). In certain particular embodiments, the polymercomprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mol % Formula(D).

Formula (E)

Formula (E) is a diradical having two points of attachment to radicalsof Formula (A). The incorporation of radicals of Formula (E) facilitatesformulation of the PBAE polymers of the invention with hydrophobiccompounds, such as PEG-lipids. Formulation with PEG-lipids is associatedwith improved particle (e.g., nanoparticle) stability and transfectionefficacy.

In certain embodiments, Formula (E) has the structure:

wherein R₉ is optionally substituted aliphatic.

In certain embodiments, Formula (E) is selected from the following:

In certain embodiments, Formula (E) has the structure:

In certain embodiments, the polymers of the invention comprise about0-50 mol % Formula (E). In certain particular embodiments, the polymersof the invention comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 mol % Formula (E).

Provided herein are certain embodiments of Formula (I), wherein:

(1) Formula (A) is selected from:

Formula (B) is selected from:

Formula (C) is selected from:

Formula (D) has a structure selected from:

and

and

Formula (E), if present, has the structure:

(2) Formula (A) has the structure:

Formula (B) has the structure:

Formula (C) has the structure:

and

Formula (D) has the structure:

(3) Formula (A) has the structure:

Formula (B) has the structure:

Formula (C) has the structure:

Formula (D) has the structure:

and

Formula (E) has the structure:

(4) Formula (A) has the structure:

Formula (B) has the structure:

Formula (C) has the structure:

and

Formula (D) has the structure:

(5) Formula (A) has the structure:

Formula (B) has the structure:

Formula (C) has the structure:

Formula (D) has the structure:

and

Formula (E) has the structure:

In embodiments (1)-(5), the polymer of Formula (I) may have a DB of 0.1,0.2, 0.3, 0.4 or 0.5. Embodiments (1), (2) and (4) may comprise radicalsof Formulae (A), (B), (C) and (D) in a molar ratio of about1:0.5:0.2:0.39; 1:0.67:0.13:0.27; 1:0.8:0.08:0.16; or 1:0.94:0:0.07.Embodiments (1), (3) and (5) may comprise radicals of Formulae (A), (B),(C), (D) and (E) in a molar ratio of about 1:0.35:0.2:0.39:0.15;1:0.47:0.13:0.27:0.2; 1:0.56:0.08:0.16:0.24; or 1:0.66:0:0.07:0.28.

Also provided herein are certain embodiments of Formula (II), wherein:

(6) Formula (A) is selected from:

and

Formula (B) is selected from:

and

Formula (D) has a structure selected from:

and

and

Formula (E), if present, has the structure:

(7) Formula (A) has the structure:

Formula (B) has the structure:

and

Formula (D) has the structure:

(8) Formula (A) has the structure:

Formula (B) has the structure:

Formula (D) has the structure:

and

Formula (E) has the structure:

(9) Formula (A) has the structure:

Formula (B) has the structure:

and

Formula (D) has the structure:

(10) Formula (A) has the structure:

Formula (B) has the structure:

Formula (D) has the structure:

and

Formula (E) has the structure:

Methods of Preparation

Provided herein is a method of preparing a polymer of Formula (I),comprising:

(i) combining a bis(acrylate) compound of Formula (A′):

with a compound of Formula (B′):

a compound of Formula (C′):

and

optionally, a compound of Formula (E′):

wherein:

R is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedarylene, optionally substituted heteroarylene, or a combination thereof;

R_(1a), R_(1b), R₂, R₃, R_(4a), and R_(4b) are each independentlyhydrogen, halogen, hydroxyl, alkoxyl, cyano, optionally substitutedaliphatic, or optionally substituted heteroaliphatic;

R₅ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedaryl, or optionally substituted heteroaryl;

R₆ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedaryl, or optionally substituted heteroaryl;

R₇ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted optionallysubstituted arylene, or optionally substituted heteroarylene;

R₉ is optionally substituted aliphatic; and

Formulae (B′), (C′) and (E′) are different from one another; and

(ii) combining the product of step (I) with a compound of Formula (D′):

wherein R₈ is optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted optionallysubstituted arylene, or optionally substituted heteroarylene; and

Formulae (B′), (C′), (D′) and (E′) are different from one another;

such that a polymer of Formula (I) is formed.

Provided herein are certain embodiments of the method, wherein:

-   -   (1) Formula (A′) has a structure selected from:

-   -   -   Formula (B′) has a structure selected from:

-   -   -    and        -   Formula (C′) has the structure:

-   -   -   Formula (D′) has a structure selected from:

-   -   (2) Formula (A′) has the structure:

-   -   -   Formula (B′) has the structure:

-   -   -   Formula (C′) has the structure:

-   -   -    and        -   Formula (D′) has the structure:

-   -   (3) Formula (A′) has the structure:

-   -   -   Formula (B′) has the structure:

-   -   -   Formula (C′) has the structure:

-   -   -    and        -   Formula (D′) has the structure:

-   -   -   Formula (E′) has the structure:

-   -   (4) Formula (A′) has the structure:

-   -   -   Formula (B′) has the structure:

-   -   -   Formula (C′) has the structure:

-   -   -    and        -   Formula (D′) has the structure:

-   -   (5) Formula (A′) has the structure:

-   -   -   Formula (B′) has the structure:

-   -   -   Formula (C′) has the structure:

-   -   -    and        -   Formula (D′) has the structure:

-   -   -   Formula (E′) has the structure:

In one aspect, provided herein is a polymer of Formula (I) preparedaccording to a method described herein, for example, Examples 2 and 4.

In certain embodiments, the monomers are reacted at temperatures rangingfrom 25 C to 90 C for 24 to 72 hours. Monomers of Formulae (A′), (B′)and/or (E′) may be added at the same time or in steps. In certainembodiments, monomers of Formulae (A′) and (C′) are reacted at 40 C for24 h (because secondary amines in the triradical are reactive at lowertemp) and then monomers of Formulae (B′) and/or (E′) added at 24 h andreacted at 90 C for a further 24-48 h. In certain embodiments, allmonomers are added at same time at a temp of 40 C for 6 h followed by anincrease in temp to 90 C and stirred up to 48 h. In other embodiments,all monomers added at same time and reacted at 90 C for 48 h.

Polymers of the invention may be prepared or used according as describedin WO 2002/031025, WO 2004/106411, and WO 2008/011561, the entirecontents of which are incorporated by reference.

Agents

Provided herein in another aspect are method of delivering an agent to acell comprising contacting the cell with a composition comprising apolymer of the invention. In certain embodiments, the method is invitro, ex vivo, or in vivo. In certain embodiments, the method is invitro or ex vivo and the cell is selected from HeLa (cervicalepithelium), A549 (lung epithelium) and C2C12 (myoblasts and myotubes).In certain embodiments, the method is in vivo and the cell is selectedfrom a muscle cell, lung epithelial cell, lung endothelial cell, spleencell and immune cell (CD45+). In certain particular embodiments, thecell is a lung cell. In certain particular embodiments, the cell is alung epithelial cell. In other particular embodiments, the cell is aspleen cell.

The agents to be delivered by polymers and compositions of the presentinvention may be therapeutic, diagnostic, or prophylactic agents. Anychemical compound to be administered to an individual may be deliveredusing the inventive polymers, compositions, complexes, picoparticles,nanoparticles, microparticles, micelles, or liposomes. The agent may bea small molecule, organometallic compound, organic compound, inorganiccompound, nucleic acid, protein, peptide, polynucleotide, metal,isotopically labeled chemical compound, drug, vaccine, immunologicalagent, etc.

In certain embodiments, the agents are organic compounds withpharmaceutical activity. In another embodiment of the invention, theagent is a clinically used drug (e.g., a drug approved in the UnitedStates by the FDA or in Europe by the EMA). In certain embodiments, thedrug is an antibiotic, anti-viral agent, anesthetic, steroidal agent,anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, etc.

In certain embodiments, the agent to be delivered is a mixture ofagents. The mixture may include 2-10 agents. For example, the mixturemay include 2, 3, 4, 5, 6, 7, 8, 9 or 10 agents.

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium. Examples of materials useful for CAT and x-ray imaging includeiodine-based materials.

Prophylactic agents include, but are not limited to, antibiotics,nutritional supplements, and vaccines. Vaccines may comprise isolatedproteins or peptides, inactivated organisms and viruses, dead organismsand viruses, genetically altered organisms or viruses, and cellextracts. Prophylactic agents may be combined with interleukins,interferon, cytokines, and adjuvants such as cholera toxin, alum,Freund's adjuvant, etc. Prophylactic agents include antigens of suchbacterial organisms as Streptococccus pneumoniae, Haemophilusinfluenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Campylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

Polynucleotide

The polynucleotide to be complexed, encapsulated by the polymers of theinvention, or included in a composition with the polymers of theinvention, may be any nucleic acid including, but not limited to, RNAand DNA.

In certain embodiments, the polynucleotide is DNA. In certain particularembodiments, the DNA is genomic DNA, synthetic DNA, a synthetic analogof DNA, cDNA or a DNA/RNA hybrid.

In certain embodiments, the polynucleotide is RNA. In certainembodiments, the polynucleotide is mRNA, siRNA, ssRNA, dsRNA, shRNA,miRNA. In certain particular embodiments, the polynucleotide is mRNA.

In certain embodiments, the polynucleotide is an RNA that carries outRNA interference (RNAi). The phenomenon of RNAi is discussed in greaterdetail, for example, in the following references, each of which isincorporated herein by reference: Elbashir et al., 2001, Genes Dev.,15:188; Fire et al., 1998, Nature, 391:806; Tabara et al., 1999, Cell,99:123; Hammond et al., Nature, 2000, 404:293; Zamore et al., 2000,Cell, 101:25; Chakraborty, 2007, Curr. Drug Targets, 8:469; and Morrisand Rossi, 2006, Gene Ther., 13:553.

In certain embodiments, the polynucleotide is a dsRNA (double-strandedRNA).

In certain embodiments, the polynucleotide is an siRNA (shortinterfering RNA).

In certain embodiments, the polynucleotide is an shRNA (short hairpinRNA).

In certain embodiments, the polynucleotide is an miRNA (micro RNA).Micro RNAs (miRNAs) are genomically encoded non-coding RNAs of about21-23 nucleotides in length that help regulate gene expression,particularly during development (see, e.g., Bartel, 2004, Cell, 116:281;Novina and Sharp, 2004, Nature, 430:161; and U.S. Patent Publication2005/0059005; also reviewed in Wang and Li, 2007, Front. Biosci.,12:3975; and Zhao, 2007, Trends Biochem. Sci., 32:189; each of which areincorporated herein by reference).

In certain embodiments, the polynucleotide is an antisense RNA.

In some embodiments, an RNA can be designed and/or predicted using oneor more of a large number of available algorithms. To give but a fewexamples, the following resources can be utilized to design and/orpredict dsRNA, siRNA, shRNA and/or miRNA: algorithms found at AlnylumOnline, Dharmacon Online, OligoEngine Online, Molecula Online, AmbionOnline, BioPredsi Online, RNAi Web Online, Chang Bioscience Online,Invitrogen Online, LentiWeb Online GenScript Online, Protocol Online;Reynolds et al., 2004, Nat. Biotechnol., 22:326; Naito et al., 2006,Nucleic Acids Res., 34:W448; Li et al., 2007, RNA, 13:1765; Yiu et al.,2005, Bioinformatics, 21:144; and Jia et al., 2006, BMC Bioinformatics,7: 271; each of which is incorporated herein by reference).

The polynucleotides may be of any size or sequence, and they may besingle- or double-stranded. In certain embodiments, the polynucleotideis greater than 100 base pairs long. In certain embodiments, thepolynucleotide is greater than 1000 base pairs long and may be greaterthan 10,000 base pairs long. The polynucleotide is optionally purifiedand substantially pure. Preferably, the polynucleotide is greater than50% pure, more preferably greater than 75% pure, and most preferablygreater than 95% pure. The polynucleotide may be provided by any meansknown in the art. In certain embodiments, the polynucleotide has beenengineered using recombinant techniques (for a more detailed descriptionof these techniques, please see Ausubel et al. Current Protocols inMolecular Biology (John Wiley & Sons, Inc., New York, 1999); MolecularCloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, andManiatis (Cold Spring Harbor Laboratory Press: 1989); each of which isincorporated herein by reference). The polynucleotide may also beobtained from natural sources and purified from contaminating componentsfound normally in nature. The polynucleotide may also be chemicallysynthesized in a laboratory. In certain embodiments, the polynucleotideis synthesized using standard solid phase chemistry.

The polynucleotide may be modified by chemical or biological means. Incertain embodiments, these modifications lead to increased stability ofthe polynucleotide. Modifications include methylation, phosphorylation,end-capping, etc.

Derivatives of polynucleotides may also be used in the presentinvention. These derivatives include modifications in the bases, sugars,and/or phosphate linkages of the polynucleotide. Modified bases include,but are not limited to, those found in the following nucleoside analogs:2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugarsinclude, but are not limited to, 2′-fluororibose, ribose,2′-deoxyribose, 3′-azido-2′,3′-dideoxyribose, 2′,3′-dideoxyribose,arabinose (the 2′-epimer of ribose), acyclic sugars, and hexoses. Thenucleosides may be strung together by linkages other than thephosphodiester linkage found in naturally occurring DNA and RNA.Modified linkages include, but are not limited to, phosphorothioate and5′-N-phosphoramidite linkages. Combinations of the various modificationsmay be used in a single polynucleotide. These modified polynucleotidesmay be provided by any means known in the art; however, as will beappreciated by those of skill in this art, the modified polynucleotidesare preferably prepared using synthetic chemistry in vitro.

The polynucleotides to be delivered may be in any form. For example, thepolynucleotide may be a circular plasmid, a linearized plasmid, acosmid, a viral genome, a modified viral genome, an artificialchromosome, etc.

The polynucleotide may be of any sequence. In certain embodiments, thepolynucleotide encodes a protein or peptide. The encoded proteins may beenzymes, structural proteins, receptors, soluble receptors, ionchannels, pharmaceutically active proteins, cytokines, interleukins,antibodies, antibody fragments, antigens, coagulation factors, albumin,growth factors, hormones, insulin, etc. The polynucleotide may alsocomprise regulatory regions to control the expression of a gene. Theseregulatory regions may include, but are not limited to, promoters,enhancer elements, repressor elements, TATA box, ribosomal bindingsites, stop site for transcription, etc. In certain embodiments, thepolynucleotide is not intended to encode a protein. For example, thepolynucleotide may be used to fix an error in the genome of the cellbeing transfected.

The polynucleotide may also be provided as an antisense agent or RNAinterference (RNAi) agent (Fire et al. Nature 391:806-811, 1998;incorporated herein by reference). Antisense therapy is meant toinclude, e.g., administration or in situ provision of single- ordouble-stranded oligonucleotides or their derivatives which specificallyhybridize, e.g., bind, under cellular conditions, with cellular mRNAand/or genomic DNA, or mutants thereof, so as to inhibit expression ofthe encoded protein, e.g., by inhibiting transcription and/ortranslation (Crooke “Molecular mechanisms of action of antisense drugs”Biochim. Biophys. Acta 1489(1):31-44, 1999; Crooke “Evaluating themechanism of action of antiproliferative antisense drugs” AntisenseNucleic Acid Drug Dev. 10(2):123-126, discussion 127, 2000; Methods inEnzymology volumes 313-314, 1999; each of which is incorporated hereinby reference). The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix (i.e., triple helix formation) (Chan et al. J. Mol. Med.75(4):267-282, 1997; incorporated herein by reference).

In certain embodiments, the polynucleotide to be delivered comprises asequence encoding an antigenic peptide or protein. Nanoparticlescontaining these polynucleotides can be delivered to an individual toinduce an immunologic response sufficient to decrease the chance of asubsequent infection and/or lessen the symptoms associated with such aninfection. The polynucleotide of these vaccines may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, alum, Freund's adjuvant, etc. A large number of adjuvantcompounds are known; a useful compendium of many such compounds isprepared by the National Institutes of Health and can be found on theinternet (www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf,incorporated herein by reference; see also Allison Dev. Biol. Stand.92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; andPhillips et al. Vaccine 10:151-158, 1992, each of which is incorporatedherein by reference).

The antigenic protein or peptides encoded by the polynucleotide may bederived from such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Campylobacter jejuni, and the like; from suchviruses as smallpox, influenza A and B, respiratory syncytial virus,parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2,cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus,papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, hepatitis A, B, C, D, and E virus, and the like; and from suchfungal, protozoan, and parasitic organisms such as Cryptococcusneoformans, Histoplasma capsulatum, Candida albicans, Candidatropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi,Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica,Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and thelike.

Polynucleotide Complexes

The ability of cationic polymers of the invention (e.g., protonatedpolymers of Formula (I)) to interact with negatively chargedpolynucleotides through electrostatic interactions is thought to atleast partially prevent the degradation of the polynucleotide. Byneutralizing the charge on the backbone of the polynucleotide, theneutral or slightly-positively-charged complex is also able to moreeasily pass through the hydrophobic membranes (e.g., cytoplasmic,lysosomal, endosomal, nuclear) of the cell. In certain embodiments, thecomplex is slightly positively charged. In certain embodiments, thecomplex has a positive ζ-potential, more preferably the ζ-potential isbetween 0 and +30.

The polymers of the present invention (e.g., polymers of Formula (I))may comprise, primary, secondary, and tertiary amines. Although theseamines are hindered, they are available to interact with apolynucleotide (e.g., DNA, RNA, synthetic analogs of DNA and/or RNA,DNA/RNA hydrids, etc.). Polynucleotides or derivatives thereof arecontacted with the polymers of the invention under conditions suitableto form polynucleotide complexes. The polymer of the invention ispreferably at least partially protonated so as to form a complex withthe negatively charged polynucleotide. In certain embodiments, thepolynucleotide complexes form particles that are useful in the deliveryof polynucleotides to cells. In certain embodiments, multiple moleculesof a polymer of the invention may be associated with a polynucleotidemolecule. The complex may include 1-100 PBAE polymers, 1-1000, 10-1000PBAE polymers, or 100-10,000 PBAE polymers.

Particles

The polymers of the present invention are useful as drug deliveryvehicles. The polymers may be used to encapsulate agents includingpolynucleotides, small molecules, proteins, peptides, metals,organometallic compounds, etc. The polymers have several properties thatmake them particularly suitable in the preparation of drug deliveryvehicles. These include: 1) the ability of the lipid to complex and“protect” labile agents; 2) the ability to buffer the pH in theendosome; 3) the ability to act as a “proton sponge” and causeendosomolytic; and/or 4) the ability to neutralize the charge onnegatively charged agents. In certain embodiments, the polymers are usedto form particles containing the agent to be delivered. These particlesmay include other materials, such as steroids (e.g., cholesterol),proteins, carbohydrates, synthetic polymers (e.g., PEG, PLGA), lipids,and natural polymers.

In certain embodiments, the particle is a microparticle or ananoparticle. In certain embodiments, the diameter of the particlesrange from between 1 micrometer to 1,000 micrometers. In certainembodiments, the diameter of the particles range from between from 1micrometer to 100 micrometers. In certain embodiments, the diameter ofthe particles range from between from 1 micrometer to 10 micrometers. Incertain embodiments, the diameter of the particles range from betweenfrom 10 micrometer to 100 micrometers. In certain embodiments, thediameter of the particles range from between from 100 micrometer to1,000 micrometers. In certain embodiments, the particles range from 1-5micrometers. In certain embodiments, the diameter of the particles rangefrom between 1 nm to 1,000 nm. In certain embodiments, the diameter ofthe particles range from between from 1 nm to 100 nm. In certainembodiments, the diameter of the particles range from between from 1 nmto 10 nm. In certain embodiments, the diameter of the particles rangefrom between from 10 nm to 100 nm. In certain embodiments, the diameterof the particles range from between from 100 nm to 1,000 nm. In certainembodiments, the particles range from 1-5 nm. In certain embodiments,the diameter of the particles range from between 1 pm to 1,000 pm. Incertain embodiments, the diameter of the particles range from betweenfrom 1 pm to 100 pm. In certain embodiments, the diameter of theparticles range from between from 1 pm to 10 pm. In certain embodiments,the diameter of the particles range from between from 10 pm to 100 pm.In certain embodiments, the diameter of the particles range from betweenfrom 100 pm to 1,000 pm. In certain embodiments, the particles rangefrom 1-5 pm.

Methods of Preparing Particles

Particles comprising polymers of the invention may be prepared using anymethod known in the art. These include, but are not limited to,lyophilization, spray drying, single and double emulsion solventevaporation, solvent extraction, phase separation, simple and complexcoacervation, and other methods well known to those of ordinary skill inthe art. In certain embodiments, methods of preparing the particles arethe double emulsion process and spray drying. The conditions used inpreparing the particles may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness”, shape, etc.). The method of preparing theparticle and the conditions (e.g., solvent, temperature, concentration,air flow rate, etc.) used may also depend on the agent beingencapsulated and/or the composition of the matrix.

Methods developed for making particles for delivery of encapsulatedagents are described in the literature (for example, please see Doubrow,M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRCPress, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release5:13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283, 1987;Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774, 1988; each of whichis incorporated herein by reference).

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, the particles can be sized, for example,using a sieve. The particle may also be coated. In certain embodiments,the particles are coated with a targeting agent. In other embodiments,the particles are coated to achieve desirable surface properties (e.g.,a particular charge).

Compositions

The present invention contemplates a polymer of the invention, e.g., apolymer of Formula (I), as a component of a composition. For example, incertain embodiments, provided is a composition comprising a polymer ofthe invention, or salt thereof, and optionally an excipient.

Compositions, as described herein, comprising a polymer of the inventionand an excipient of some sort may be useful in a variety of medical andnon-medical applications. For example, pharmaceutical compositionscomprising a polymer of the invention and an excipient may be useful inthe delivery of an effective amount of an agent to a subject in needthereof. Nutraceutical compositions comprising a polymer of theinvention and an excipient may be useful in the delivery of an effectiveamount of a nutraceutical, e.g., a dietary supplement, to a subject inneed thereof. Cosmetic compositions comprising a polymer of theinvention and an excipient may be formulated as a cream, ointment, balm,paste, film, or liquid, etc., and may be useful in the application ofmake-up, hair products, and materials useful for personal hygiene, etc.Compositions comprising a polymer of the invention and an excipient maybe useful for non-medical applications, e.g., such as an emulsion oremulsifier, useful, for example, as a food component, for extinguishingfires, for disinfecting surfaces, for oil cleanup, etc.

Peptides play significant roles in endogenous cellular signaling andtrafficking pathways, and offer tremendous potential in leveraging suchinteractions to enhance the delivery efficiency of systems whichincorporate peptide moieties. Thus, compositions comprising a polymer ofthe invention and an excipient may further be useful in bioprocessing,such as a cell's bioprocessing of a commercially useful chemical orfuel. For example, intracellular delivery of the polymer of theinvention or an agent complexed thereto may be useful in bioprocessingby maintaining the cell's health and/or growth, e.g., in themanufacturing of proteins.

The composition may comprise one type of polymer of the invention butmay also comprise any number of different types, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more different types of polymers of the invention.

Accordingly, provided herein in another aspect is a compositioncomprising a polymer of the invention, e.g., a polymer of Formula (I).In certain embodiments, the composition is a cosmetic composition. Incertain embodiments, the composition is a pharmaceutical composition andfurther comprises a pharmaceutically acceptable carrier.

The composition may comprise an agent, as described herein. When theagent is a polynucleotide, the composition may be characterized in termsof an N/P ratio (i.e., the ratio of moles of the amine groups of thepolymer of the invention to moles of the phosphate groups of thepolynucleotide). In certain embodiments, the N/P ratio of compositionsof the invention is in the range of 1-70, 5-60, 5-50, 10-40, or 20-30.

In certain embodiments, the composition is formulated for intravenousdelivery. In certain embodiments, the composition is formulated foraerosol delivery. In certain embodiments, the composition is in the formof a particle.

In certain embodiments, the particle is produced by lyophilization. Incertain embodiments, the lyophilized particle comprises a carbohydrateadditive. In certain embodiments, the carbohydrate is a non-reducingsugar. In certain particular embodiments, the non-reducing sugar issucrose. In certain embodiments, the lyophilized particle is retains atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90% efficacy after storage for 14-90 days at −80° C. Incertain embodiments, the composition is formulated at a pH of about 5.2to about 7.4.

Pharmaceutical Compositions

Once the complexes, micelles, liposomes, or particles have beenprepared, they may be combined with one or more pharmaceuticalexcipients to form a pharmaceutical composition that is suitable toadminister to animals including humans. As would be appreciated by oneof skill in this art, the excipients may be chosen based on the route ofadministration as described below, the agent being delivered, timecourse of delivery of the agent, etc.

Pharmaceutical compositions of the present invention and for use inaccordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose, and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; detergentssuch as Tween 80; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toanimals, orally, rectally, parenterally, intracisternally,intravaginally, intranasally, intraperitoneally, topically (as bypowders, creams, ointments, or drops), bucally, or as an oral or nasalspray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e.,microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipidcomplexes), the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Incertain embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the particles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

Targeting Agents

The inventive polymer, compositions, complexes, liposomes, micelles,microparticles, picoparticles, and nanoparticles may be modified toinclude targeting agents since it is often desirable to target aparticular cell, collection of cells, or tissue. A variety of targetingagents that direct pharmaceutical compositions to particular cells areknown in the art (see, for example, Cotten et al., Methods Enzym.217:618, 1993; incorporated herein by reference). The targeting agentsmay be included throughout the particle or may be only on the surface.The targeting agent may be a protein, peptide, carbohydrate,glycoprotein, lipid, small molecule, nucleic acid, etc. The targetingagent may be used to target specific cells or tissues or may be used topromote endocytosis or phagocytosis of the particle. Examples oftargeting agents include, but are not limited to, antibodies, fragmentsof antibodies, low-density lipoproteins (LDLs), transferrin,asialycoproteins, gp120 envelope protein of the human immunodeficiencyvirus (HIV), carbohydrates, receptor ligands, sialic acid, etc. If thetargeting agent is included throughout the particle, the targeting agentmay be included in the mixture that is used to form the particles. Ifthe targeting agent is only on the surface, the targeting agent may beassociated with (i.e., by covalent, hydrophobic, hydrogen bonding, vander Waals, or other interactions) the formed particles using standardchemical techniques.

Treatment Methods

It is estimated that over 10,000 human diseases are caused by geneticdisorders, which are abnormalities in genes or chromosomes. See, e.g.,McClellan, J. and M. C. King, Genetic heterogeneity in human disease.Cell. 141(2): p. 210-7; Leachman, S. A., et al., Therapeutic siRNAs fordominant genetic skin disorders including pachyonychia congenita. JDermatol Sci, 2008. 51(3): p. 151-7. Many of these diseases are fatal,such as cancer, severe hypercholesterolemia, and familial amyloidoticpolyneuropathy. See, e.g., Frank-Kamenetsky, M., et al., TherapeuticRNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents andLDL cholesterol in nonhuman primates. Proc Natl Acad Sci USA, 2008.105(33): p. 11915-20; Coelho, T., Familial amyloid polyneuropathy: newdevelopments in genetics and treatment. Curr Opin Neurol, 1996. 9(5): p.355-9. Since the discovery of gene expression silencing via RNAinterference (RNAi) by Fire and Mello (Fire, A., et al., Potent andspecific genetic interference by double-stranded RNA in Caenorhabditiselegans. Nature, 1998. 391(6669): p. 806-11), there has been extensiveeffort toward developing therapeutic applications for RNAi in humans.See, e.g., Davis, M. E., The first targeted delivery of siRNA in humansvia a self-assembling, cyclodextrin polymer-based nanoparticle: fromconcept to clinic. Mol Pharm, 2009. 6(3): p. 659-68; Whitehead, K. A.,R. Langer, and D. G. Anderson, Knocking down barriers: advances in siRNAdelivery. Nat. Rev. Drug Discovery, 2009. 8(2): p. 129-138; Tan, S. J.,et al., Engineering Nanocarriers for siRNA Delivery. Small. 7(7): p.841-56; Castanotto, D. and J. J. Rossi, The promises and pitfalls ofRNA-interference-based therapeutics. Nature, 2009. 457(7228): p. 426-33;Chen, Y. and L. Huang, Tumor-targeted delivery of siRNA by non-viralvector: safe and effective cancer therapy. Expert Opin Drug Deliv, 2008.5(12): p. 1301-11; Weinstein, S. and D. Peer, RNAi nanomedicines:challenges and opportunities within the immune system. Nanotechnology.21(23): p. 232001; Fenske, D. B. and P. R. Cullis, Liposomalnanomedicines. Expert Opin Drug Deliv, 2008. 5(1): p. 25-44; and Thiel,K. W. and P. H. Giangrande, Therapeutic applications of DNA and RNAaptamers. Oligonucleotides, 2009. 19(3): p. 209-22. Currently, there aremore than 20 clinical trials ongoing or completed involving siRNAtherapeutics, which have shown promising results for the treatment ofvarious diseases. See, e.g., Burnett, J. C., J. J. Rossi, and K.Tiemann, Current progress of siRNA/shRNA therapeutics in clinicaltrials. Biotechnol J. 6(9): p. 1130-46. However, the efficient and safedelivery of siRNA is still a key challenge in the development of siRNAtherapeutics. See, e.g., Juliano, R., et al., Biological barriers totherapy with antisense and siRNA oligonucleotides. Mol Pharm, 2009.6(3): p. 686-95.

Accordingly, provided herein are methods of using polymers of theinvention, e.g., a polymer of Formula (I), for the treatment of adisease, disorder, or condition from which a subject suffers. It iscontemplated that polymers of the invention will be useful in thetreatment of a variety of diseases, disorders, or conditions, especiallya system for delivering agents useful in the treatment of thatparticular disease, disorder, or condition. “Disease,” “disorder,” and“condition” are used interchangeably herein. In certain embodiments, thedisease, disorder or condition from which a subject suffers is caused byan abnormality in a gene or chromosome of the subject.

For example, in one embodiment, provided is a method of treatingdisease, disorder, or condition from which a subject suffers, comprisingadministering to a subject in need thereof an effective amount of acomposition comprising a polymer of the invention, e.g., a polymer ofFormula (I), or salt thereof. Exemplary disease, disorder, or conditionscontemplated include, but are not limited to, proliferative disorders,inflammatory disorders, autoimmune disorders, painful conditions, lungdiseases, liver diseases, amyloid neuropathies, enzyme deficiencies andcystic fibrosis.

In certain particular embodiments, the method is for treating lungdisease. In certain embodiments, the lung disease is asthma, chronicobstructive pulmonary disease (COPD), chronic bronchitis, emphysema,pulmonary hypertension, pulmonary fibrosis (e.g., idiopathic pulmonaryfibrosis, fibrotic interstitial lung disease, interstitial pneumonia,fibrotic variant of non-specific interstitial pneumonia, or cysticfibrosis), sarcoidosis, influenza, pneumonia, tuberculosis, or lungcancer. In certain embodiments, the lung cancer is bronchogeniccarcinoma, small cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), or adenocarcinoma of the lung

In certain embodiments, the composition further comprises, in additionto the polymer of the invention, a therapeutic agent useful in treatingthe disease, disorder, or condition. In certain embodiments, the polymerof the invention encapsulates the other (therapeutic) agent. In certainembodiments, the polymer of the invention and the other (therapeutic)agent form a particle (e.g., a nanoparticle, a microparticle, a micelle,a liposome, a lipoplex).

In certain embodiments, the condition is a proliferative disorder and,in certain embodiments, the composition further includes an anti-canceragent. Exemplary proliferative diseases include, but are not limited to,tumors, benign neoplasms, pre-malignant neoplasms (carcinoma in situ),and malignant neoplasms (cancers).

Exemplary cancers include, but are not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing's sarcoma, eye cancer (e.g., intraocular melanoma,retinoblastoma), familiar hypereosinophilia, gall bladder cancer,gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromaltumor (GIST), head and neck cancer (e.g., head and neck squamous cellcarcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC),throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngealcancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemiasuch as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL),acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., “Waldenström's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), leiomyosarcoma (LMS), mastocytosis (e.g., systemicmastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocyticleukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilicsyndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g.,gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoidtumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma,pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer(e.g., Paget's disease of the penis and scrotum), pinealoma, primitiveneuroectodermal tumor (PNT), prostate cancer (e.g., prostateadenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer,skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g.,appendix cancer), soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous glandcarcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g.,seminoma, testicular embryonal carcinoma), thyroid cancer (e.g.,papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC),medullary thyroid cancer), urethral cancer, vaginal cancer and vulvarcancer (e.g., Paget's disease of the vulva).

Anti-cancer agents encompass biotherapeutic anti-cancer agents as wellas chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limitedto, interferons, cytokines (e.g., tumor necrosis factor, interferon α,interferon γ), vaccines, hematopoietic growth factors, monoclonalserotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1,2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) andantibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab),ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR(tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to,anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRHagonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamideand bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A(2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide,trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas(e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide),platinum containing compounds (e.g. cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine,and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalentsuch as nanoparticle albumin-bound paclitaxel (ABRAXANE),docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin),polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex,CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxelbound to the erbB2-recognizing peptide EC-1), and glucose-conjugatedpaclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline(e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDRinhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin),imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g.,axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib (IRESSA®)), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474),vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab(AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab(VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib(NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumabozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765,AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523,PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/orXL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTORinhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus(RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235(Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502(Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)),oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed,cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,aminopterin, and hexamethyl melamine.

In certain embodiments, the condition is an inflammatory disorder and,in certain embodiments, the composition further includes ananti-inflammatory agent. The term “inflammatory disorder” refers tothose diseases, disorders or conditions that are characterized by signsof pain (dolor, from the generation of noxious substances and thestimulation of nerves), heat (calor, from vasodilatation), redness(rubor, from vasodilatation and increased blood flow), swelling (tumor,from excessive inflow or restricted outflow of fluid), and/or loss offunction (functio laesa, which can be partial or complete, temporary orpermanent Inflammation takes on many forms and includes, but is notlimited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic,diffuse, disseminated, exudative, fibrinous, fibrosing, focal,granulomatous, hyperplastic, hypertrophic, interstitial, metastatic,necrotic, obliterative, parenchymatous, plastic, productive,proliferous, pseudomembranous, purulent, sclerosing, seroplastic,serous, simple, specific, subacute, suppurative, toxic, traumatic,and/or ulcerative inflammation.

Exemplary inflammatory disorders include, but are not limited to,inflammation associated with acne, anemia (e.g., aplastic anemia,haemolytic autoimmune anaemia), asthma, arteritis (e.g., polyarteritis,temporal arteritis, periarteritis nodosa, Takayasu's arteritis),arthritis (e.g., crystalline arthritis, osteoarthritis, psoriaticarthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis andReiter's arthritis), ankylosing spondylitis, amylosis, amyotrophiclateral sclerosis, autoimmune diseases, allergies or allergic reactions,atherosclerosis, bronchitis, bursitis, chronic prostatitis,conjunctivitis, Chagas disease, chronic obstructive pulmonary disease,cermatomyositis, diverticulitis, diabetes (e.g., type I diabetesmellitus, type 2 diabetes mellitus), a skin condition (e.g., psoriasis,eczema, burns, dermatitis, pruritus (itch)), endometriosis,Guillain-Barre syndrome, infection, ischaemic heart disease, Kawasakidisease, glomerulonephritis, gingivitis, hypersensitivity, headaches(e.g., migraine headaches, tension headaches), ileus (e.g.,postoperative ileus and ileus during sepsis), idiopathicthrombocytopenic purpura, interstitial cystitis (painful bladdersyndrome), gastrointestinal disorder (e.g., selected from peptic ulcers,regional enteritis, diverticulitis, gastrointestinal bleeding,eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis,eosinophilic gastritis, eosinophilic gastroenteritis, eosinophiliccolitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, orits synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn'sdisease, ulcerative colitis, collagenous colitis, lymphocytic colitis,ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminatecolitis) and inflammatory bowel syndrome (IBS)), lupus, multiplesclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephroticsyndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers,polymyositis, primary biliary cirrhosis, neuroinflammation associatedwith brain disorders (e.g., Parkinson's disease, Huntington's disease,and Alzheimer's disease), prostatitis, chronic inflammation associatedwith cranial radiation injury, pelvic inflammatory disease, reperfusioninjury, regional enteritis, rheumatic fever, systemic lupuserythematosus, schleroderma, scierodoma, sarcoidosis,spondyloarthopathies, Sjogren's syndrome, thyroiditis, transplantationrejection, tendonitis, trauma or injury (e.g., frostbite, chemicalirritants, toxins, scarring, burns, physical injury), vasculitis,vitiligo and Wegener's granulomatosis.

In certain embodiments, the inflammatory disorder is inflammationassociated with a proliferative disorder, e.g., inflammation associatedwith cancer.

In certain embodiments, the condition is an autoimmune disorder and, incertain embodiments, the composition further includes animmunomodulatory agent. Exemplary autoimmune disorders include, but arenot limited to, arthritis (including rheumatoid arthritis,spondyloarthopathies, gouty arthritis, degenerative joint diseases suchas osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome,ankylosing spondylitis, undifferentiated spondylitis, Behcet's disease,haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateralsclerosis, amylosis, acute painful shoulder, psoriatic, and juvenilearthritis), asthma, atherosclerosis, osteoporosis, bronchitis,tendonitis, bursitis, skin condition (e.g., psoriasis, eczema, burns,dermatitis, pruritus (itch)), enuresis, eosinophilic disease,gastrointestinal disorder (e.g., selected from peptic ulcers, regionalenteritis, diverticulitis, gastrointestinal bleeding, eosinophilicgastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilicgastritis, eosinophilic gastroenteritis, eosinophilic colitis),gastritis, diarrhea, gastroesophageal reflux disease (GORD, or itssynonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease,ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemiccolitis, diversion colitis, Behcet's syndrome, indeterminate colitis)and inflammatory bowel syndrome (IBS)), and disorders ameliorated by agastroprokinetic agent (e.g., ileus, postoperative ileus and ileusduring sepsis; gastroesophageal reflux disease (GORD, or its synonymGERD); eosinophilic esophagitis, gastroparesis such as diabeticgastroparesis; food intolerances and food allergies and other functionalbowel disorders, such as non-ulcerative dyspepsia (NUD) and non-cardiacchest pain (NCCP, including costo-chondritis)).

In certain embodiments, the condition is a painful condition and, incertain embodiments, the composition further includes an analgesicagent. A “painful condition” includes, but is not limited to,neuropathic pain (e.g., peripheral neuropathic pain), central pain,deafferentiation pain, chronic pain (e.g., chronic nociceptive pain, andother forms of chronic pain such as post-operative pain, e.g., painarising after hip, knee, or other replacement surgery), pre-operativepain, stimulus of nociceptive receptors (nociceptive pain), acute pain(e.g., phantom and transient acute pain), noninflammatory pain,inflammatory pain, pain associated with cancer, wound pain, burn pain,postoperative pain, pain associated with medical procedures, painresulting from pruritus, painful bladder syndrome, pain associated withpremenstrual dysphoric disorder and/or premenstrual syndrome, painassociated with chronic fatigue syndrome, pain associated with pre-termlabor, pain associated with withdrawal symptoms from drug addiction,joint pain, arthritic pain (e.g., pain associated with crystallinearthritis, osteoarthritis, psoriatic arthritis, gouty arthritis,reactive arthritis, rheumatoid arthritis or Reiter's arthritis),lumbosacral pain, musculo-skeletal pain, headache, migraine, muscleache, lower back pain, neck pain, toothache, dental/maxillofacial pain,visceral pain and the like. One or more of the painful conditionscontemplated herein can comprise mixtures of various types of painprovided above and herein (e.g. nociceptive pain, inflammatory pain,neuropathic pain, etc.). In some embodiments, a particular pain candominate. In other embodiments, the painful condition comprises two ormore types of pains without one dominating. A skilled clinician candetermine the dosage to achieve a therapeutically effective amount for aparticular subject based on the painful condition.

In certain embodiments, the painful condition is inflammatory pain. Incertain embodiments, the painful condition (e.g., inflammatory pain) isassociated with an inflammatory disorder and/or an autoimmune disorder.

In certain embodiments, the condition is a liver disease and, in certainembodiments, the composition further includes an agent useful intreating liver disease. Exemplary liver diseases include, but are notlimited to, drug-induced liver injury (e.g., acetaminophen-induced liverinjury), hepatitis (e.g., chronic hepatitis, viral hepatitis,alcohol-induced hepatitis, autoimmune hepatitis, steatohepatitis),non-alcoholic fatty liver disease, alcohol-induced liver disease (e.g.,alcoholic fatty liver, alcoholic hepatitis, alcohol-related cirrhosis),hypercholesterolemia (e.g., severe hypercholesterolemia),transthyretin-related hereditary amyloidosis, liver cirrhosis, livercancer, primary biliary cirrhosis, cholestatis, cystic disease of theliver, and primary sclerosing cholangitis. In certain embodiments theliver disease is associated with inflammation.

In certain embodiments, the condition is a familial amyloid neuropathyand, in certain embodiments, the composition further includes an agentuseful in a familial amyloid neuropathy.

Compositions comprising a polymer of the invention may be administeredin such amounts, time, and route deemed necessary in order to achievethe desired result. The exact amount of the active ingredient will varyfrom subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the infection, the particularactive ingredient, its mode of administration, its mode of activity, andthe like. Compositions are preferably formulated in dosage unit form forease of administration and uniformity of dosage. It will be understood,however, that the total daily usage of the active ingredient will bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective dose level for anyparticular subject will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe active ingredient employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific active ingredient employed; the duration of the treatment;drugs used in combination or coincidental with the specific activeingredient employed; and like factors well known in the medical arts.

Kits

The invention also provides kits for use in preparing the polymers ofthe invention, or compositions thereof. The kit may include any or allof the following: amines of Formulae (B′), (C′), (D′), and/or (E′),diacrylates of Formula (A′), a combination of said amines anddiacrylates, poly(beta-amino esters), vials, solvent, buffers,multi-well plates, salts, agents as described herein (e.g.,polynucleotides, proteins, or small molecules) and instructions. Theinstructions include ways of preparing the inventive end-modifiedpolymers with various properties. In certain embodiments, the kit istailored for preparation of end-modified polymers with a desiredproperty or for a desired use. In certain embodiments, the kit includesall the items necessary to prepare one or more polymers of theinvention, or compositions thereof.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds and polymers described herein can comprise one or moreasymmetric centers, and thus can exist in various stereoisomeric forms,e.g., enantiomers and/or diastereomers. For example, the polymersdescribed herein can be in the form of an individual enantiomer,diastereomer or geometric isomer, or can be in the form of a mixture ofstereoisomers, including racemic mixtures and mixtures enriched in oneor more stereoisomer. Isomers can be isolated from mixtures by methodsknown to those skilled in the art, including chiral high pressure liquidchromatography (HPLC) and the formation and crystallization of chiralsalts; or preferred isomers can be prepared by asymmetric syntheses.See, for example, Jacques et al., Enantiomers, Racemates and Resolutions(Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725(1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses polymers asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

Unless otherwise stated, structures depicted herein are also meant toinclude polymers that differ only in the presence of one or moreisotopically enriched atoms. For example, polymers having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of a carbon bya ¹³C- or ¹⁴C-enriched carbon are within the scope of the disclosure.Such polymers are useful, for example, as analytical tools or probes inbiological assays.

When a range of values is listed, it is intended to encompass each valueand subrange within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic” refers to alkyl, alkenyl, alkynyl, and carbocyclicgroups. Likewise, the term “heteroaliphatic” refers to heteroalkyl,heteroalkenyl, heteroalkynyl, and heterocyclic groups.

The term “alkyl” refers to a radical of a straight-chain or branchedsaturated hydrocarbon group having from 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), propyl(C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl,sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl,neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C₆) (e.g.,2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,and n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇),n-octyl (C₈), and the like. Unless otherwise specified, each instance ofan alkyl group is independently unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents(e.g., halogen, such as F). In certain embodiments, the alkyl group isan unsubstituted C₁₋₁₀ alkyl (such as unsubstituted C₁₋₆ alkyl, e.g.,—CH₃ (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g.,unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)),unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu),unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl(sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certainembodiments, the alkyl group is a substituted C₁₋₁₀ alkyl (such assubstituted C₁₋₆ alkyl, e.g., —CH₂F, —CHF₂, —CF₃ or benzyl (Bn)).

The term “alkoxy” refers to a radical of the formula —O(alkyl).

The term “carbamoyl” refers to a radical of formula —O(C═O)NR₂ orformula —N(R)(C═O)OR, wherein each R independently is hydrogen,optionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted optionally substitutedaryl, or optionally substituted heteroaryl.

The term “ureido” refers to a radical of the formula —N(R)(C═O)NR₂,wherein each R independently is hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted optionally substituted aryl, or optionally substitutedheteroaryl.

The term “amido” refers to a radical of the formula —(C═O)NR₂ or formula—N(R)(C═O)R, wherein each R independently is hydrogen, optionallysubstituted aliphatic, optionally substituted heteroaliphatic,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted optionally substituted aryl, or optionallysubstituted heteroaryl.

The term “carbonyldioxyl” refers to a radical of the formula —O(C═O)OR,wherein each R independently is hydrogen, optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted optionally substituted aryl, or optionally substitutedheteroaryl.

The term “alkylthioether” refers to a radical of the formula —R—S—R,wherein each R independently is optionally substituted alkyl.

The term “haloalkyl” is a substituted alkyl group, wherein one or moreof the hydrogen atoms are independently replaced by a halogen, e.g.,fluoro, bromo, chloro, or iodo. “Perhaloalkyl” is a subset of haloalkyl,and refers to an alkyl group wherein all of the hydrogen atoms areindependently replaced by a halogen, e.g., fluoro, bromo, chloro, oriodo. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms(“C₁₋₈ haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 6carbon atoms (“C₁₋₆ haloalkyl”). In some embodiments, the haloalkylmoiety has 1 to 4 carbon atoms (“C₁₋₄ haloalkyl”). In some embodiments,the haloalkyl moiety has 1 to 3 carbon atoms (“C₁₋₃ haloalkyl”). In someembodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C₁₋₂haloalkyl”). In some embodiments, all of the haloalkyl hydrogen atomsare replaced with fluoro to provide a perfluoroalkyl group. In someembodiments, all of the haloalkyl hydrogen atoms are replaced withchloro to provide a “perchloroalkyl” group. Examples of haloalkyl groupsinclude —CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

The term “heteroalkyl” refers to an alkyl group, which further includesat least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected fromoxygen, nitrogen, or sulfur within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkyl group refers toa saturated group having from 1 to 10 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms andfor 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₁₀ alkyl.

The term “alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In someembodiments, an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉ alkenyl”).In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C₂₋₈alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms(“C₂₋₇ alkenyl”). In some embodiments, an alkenyl group has 2 to 6carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, an alkenyl group has2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, an alkenylgroup has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In some embodiments, analkenyl group has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”). In someembodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”). The oneor more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of C₂₋₄ alkenylgroups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl(C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkenyl groups as well aspentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additionalexamples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl(C₈), and the like. Unless otherwise specified, each instance of analkenyl group is independently unsubstituted (an “unsubstitutedalkenyl”) or substituted (a “substituted alkenyl”) with one or moresubstituents. In certain embodiments, the alkenyl group is anunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis a substituted C₂₋₁₀ alkenyl. In an alkenyl group, a C═C double bondfor which the stereochemistry is not specified (e.g., —CH═CHCH₃ or

may be in the (E)- or (Z)-configuration.

The term “heteroalkenyl” refers to an alkenyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkenylgroup refers to a group having from 2 to 10 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has2 to 9 carbon atoms at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 8 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbonatoms, at least one double bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and for 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”). Insome embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

The term “alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C₂₋₁₀alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms(“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has 2 to 8carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynyl group has2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, an alkynylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In some embodiments, analkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”). In someembodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄ alkynyl”).In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C₂₋₃alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂alkynyl”). The one or more carbon-carbon triple bonds can be internal(such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples ofC₂₋₄ alkynyl groups include, without limitation, ethynyl (C₂),1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), andthe like. Examples of C₂₋₆ alkenyl groups include the aforementionedC₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and thelike. Additional examples of alkynyl include heptynyl (C₇), octynyl(C₈), and the like. Unless otherwise specified, each instance of analkynyl group is independently unsubstituted (an “unsubstitutedalkynyl”) or substituted (a “substituted alkynyl”) with one or moresubstituents. In certain embodiments, the alkynyl group is anunsubstituted C₂₋₁₀ alkynyl. In certain embodiments, the alkynyl groupis a substituted C₂₋₁₀ alkynyl.

The term “heteroalkynyl” refers to an alkynyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkynylgroup refers to a group having from 2 to 10 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbonatoms, at least one triple bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and for 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”). Insome embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₁₀ alkynyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturated radicalhaving from 3 to 14 ring carbon atoms (“C₃₋₁₄ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 7 ring carbon atoms (“C₃₋₇ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₆). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl. In certain embodiments, thecarbocyclyl includes 0, 1, or 2 C═C double bonds in the carbocyclic ringsystem, as valency permits.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl. In certain embodiments, the heterocyclyl issubstituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl,wherein 1, 2, or 3 atoms in the heterocyclic ring system areindependently oxygen, nitrogen, or sulfur, as valency permits.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, dioxolanyl,oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groupscontaining 3 heteroatoms include, without limitation, triazolinyl,oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclylgroups containing 1 heteroatom include, without limitation, piperidinyl,tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-memberedheterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary6-membered heterocyclyl groups containing 3 heteroatoms include, withoutlimitation, triazinyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14 π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

“Aralkyl” is a subset of “alkyl” and refers to an alkyl groupsubstituted by an aryl group, wherein the point of attachment is on thealkyl moiety.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6, 10, or 14 π electrons shared in a cyclic array) havingring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groupsthat contain one or more nitrogen atoms, the point of attachment can bea carbon or nitrogen atom, as valency permits. Heteroaryl polycyclicring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the point of attachment is on the heteroaryl ring, and insuch instances, the number of ring members continue to designate thenumber of ring members in the heteroaryl ring system. “Heteroaryl” alsoincludes ring systems wherein the heteroaryl ring, as defined above, isfused with one or more aryl groups wherein the point of attachment iseither on the aryl or heteroaryl ring, and in such instances, the numberof ring members designates the number of ring members in the fusedpolycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groupswherein one ring does not contain a heteroatom (e.g., indolyl,quinolinyl, carbazolyl, and the like) the point of attachment can be oneither ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl). In certain embodiments, the heteroaryl is substituted orunsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3,or 4 atoms in the heteroaryl ring system are independently oxygen,nitrogen, or sulfur. In certain embodiments, the heteroaryl issubstituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl,wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system areindependently oxygen, nitrogen, or sulfur.

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

The term “unsaturated bond” refers to a double or triple bond.

The term “unsaturated” or “partially unsaturated” refers to a moietythat includes at least one double or triple bond.

The term “saturated” refers to a moiety that does not contain a doubleor triple bond, i.e., the moiety only contains single bonds.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

Groups recited herein in variable definitions are optionally substitutedunless expressly provided otherwise. The term “optionally substituted”refers to being substituted or unsubstituted. In certain embodiments,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl groups are optionally substituted. “Optionally substituted”refers to a group which may be substituted or unsubstituted (e.g.,“substituted” or “unsubstituted” aliphatic, “substituted” or“unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl,“substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroaliphatic, “substituted” or “unsubstituted”heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl,“substituted” or “unsubstituted” heteroalkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”means that at least one hydrogen present on a group is replaced with apermissible substituent, e.g., a substituent which upon substitutionresults in a stable polymer, e.g., a polymer which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, and includes any of the substituents described herein thatresults in the formation of a stable polymer. The present inventioncontemplates any and all such combinations in order to arrive at astable polymer. For purposes of this invention, heteroatoms such asnitrogen may have hydrogen substituents and/or any suitable substituentas described herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety. The invention is notintended to be limited in any manner by the exemplary substituentsdescribed herein.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(aa))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl,C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(aa)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)groups;each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, heteroC₁₋₁₀alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(bb) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(cc) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups;each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two geminalR^(dd) substituents can be joined to form ═O or ═S;each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆ alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl,3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein eachalkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or two R^(ff)groups are joined to form a 3-10 membered heterocyclyl or 5-10 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups; andeach instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl),—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl), C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl), —OP(═O)(OC₁₋₆ alkyl)₂,C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

In certain embodiments, the carbon atom substituents are independentlyhalogen, substituted or unsubstituted C₁₋₆ alkyl, —OR^(aa), —SR^(aa),—N(R^(bb))₂, —CN, —SCN, —NO₂, —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), or —NR^(bb)C(═O)N(R^(bb))₂. Incertain embodiments, the carbon atom substituents are independentlyhalogen, substituted or unsubstituted C₁₋₆ alkyl, —OR^(aa), —SR^(aa),—N(R^(bb))₂, —CN, —SCN, or —NO₂.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine(chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “-hydroxyl” or “—OH” refers to the group —OH. The term“substituted hydroxyl” or “substituted —OH,” by extension, refers to ahydroxyl group wherein the oxygen atom directly attached to the parentmolecule is substituted with a group other than hydrogen, and includesgroups selected from —OR^(aa), —ON(R^(bb))₂, —OC(═O)SR^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂, —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —OC(═NR^(bb))N(R^(bb))₂, —OS(═O)R^(aa),—OSO₂R^(aa), —OSi(R^(aa))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —OP(═O)₂R^(aa),—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —OP(═O)₂N(R^(bb))₂, and—OP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein.

The term “amino” refers to the group —NH₂. The term “substituted amino,”by extension, refers to a monosubstituted amino, a disubstituted amino,or a trisubstituted amino. In certain embodiments, the “substitutedamino” is a monosubstituted amino or a disubstituted amino group.

The term “monosubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith one hydrogen and one group other than hydrogen, and includes groupsselected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) andR^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

The term “disubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith two groups other than hydrogen, and includes groups selected from—N(R^(bb))₂, —NR^(bb) C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂,wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with theproviso that the nitrogen atom directly attached to the parent moleculeis not substituted with hydrogen.

The term “trisubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith three groups, and includes groups selected from —N(R^(bb))₃ and—N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as defined herein.

The term “carbonyl” refers a group wherein the carbon directly attachedto the parent molecule is sp² hybridized, and is substituted with anoxygen, nitrogen or sulfur atom, e.g., a group selected from ketones(—C(═O)R^(aa)), carboxylic acids (—CO₂H), carboxylates (—CO₂), aldehydes(—CHO), esters (—CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)), amides(—C(═O)N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂), and imines(—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa)), —C(═NR^(bb))N(R^(bb))₂),wherein R^(aa) and R^(bb) are as defined herein.

The term “oxo” refers to the group ═O, and the term “thiooxo” refers tothe group ═S.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substituents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to an N atom are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, andwherein R^(aa), R^(bb), R^(cc) and R^(dd) are as defined above.

The following definitions are more general terms used throughout thepresent application.

The term “pharmaceutically acceptable salt” refers to those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response, and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the polymers of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acids,such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid, and perchloric acid or with organic acids, such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, ormalonic acid or by using other methods known in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide, 2-OH-ethanesulfonate,lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oleate, oxalate, palmitate, pamoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, valerate salts, and the like. Salts derived fromappropriate bases include alkali metal, alkaline earth metal, ammonium,and N⁺(C₁₋₄ alkyl)₄ ⁻ salts. Representative alkali or alkaline earthmetal salts include sodium, lithium, potassium, calcium, magnesium, andthe like. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

In certain embodiments, the polymer of Formula (I) is a salt. In certainparticular embodiments, the polymer of Formula (I) is a pharmaceuticallyacceptable salt.

In certain embodiments, one or more radicals of Formulae (A), (B), (C),(D), (E), (A′), (B′), (C′), (D′), and (E′) are salts. In certainparticular embodiments, one or more radicals of Formulae (A), (B), (C),(D), (E), (A′), (B′), (C′), (D′), and (E′) are pharmaceuticallyacceptable salts.

The terms “composition” and “formulation” are used interchangeably.

As used herein, the term “polyplex” refers to a complex comprising apolymer of the invention and one or more agents. In certain embodiments,a polyplex takes the form of a particle, such as a nanoparticle.

A “subject” to which administration is contemplated refers to a human(i.e., male or female of any age group, e.g., pediatric subject (e.g.,infant, child, or adolescent) or adult subject (e.g., young adult,middle-aged adult, or senior adult)) or non-human animal. In certainembodiments, the non-human animal is a mammal (e.g., primate (e.g.,cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g.,cattle, cows, pig, horse, sheep, goat, cat, or dog), or bird (e.g.,commercially relevant bird, such as chicken, duck, goose, or turkey)).In certain embodiments, the non-human animal is a fish, reptile, oramphibian. The non-human animal may be a male or female at any stage ofdevelopment. The non-human animal may be a transgenic animal orgenetically engineered animal. A “patient” refers to a human subject inneed of treatment of a disease. The subject may also be a plant. Incertain embodiments, the plant is a land plant. In certain embodiments,the plant is a non-vascular land plant. In certain embodiments, theplant is a vascular land plant. In certain embodiments, the plant is aseed plant. In certain embodiments, the plant is a cultivated plant. Incertain embodiments, the plant is a dicot. In certain embodiments, theplant is a monocot. In certain embodiments, the plant is a floweringplant. In some embodiments, the plant is a cereal plant, e.g., maize,corn, wheat, rice, oat, barley, rye, or millet. In some embodiments, theplant is a legume, e.g., a bean plant, e.g., soybean plant. In someembodiments, the plant produces fruit. In some embodiments, the plant isa tree or shrub.

The term “administer,” “administering,” or “administration” refers toimplanting, absorbing, ingesting, injecting, inhaling, or otherwiseintroducing a polymer described herein, or a composition thereof, in oron a subject.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, delaying the onset of, or inhibiting the progress of adisease (e.g., a bacterial infection) described herein. In someembodiments, treatment may be administered after one or more signs orsymptoms of the disease have developed or have been observed. In otherembodiments, treatment may be administered in the absence of signs orsymptoms of the disease. For example, treatment may be administered to asusceptible subject prior to the onset of symptoms (e.g., in light of ahistory of symptoms and/or in light of exposure to a pathogen).Treatment may also be continued after symptoms have resolved, forexample, to delay and/or prevent recurrence.

The term “prevent,” “preventing,” or “prevention” refers to aprophylactic treatment of a subject who is not and was not with adisease (e.g., a bacterial infection) but is at risk of developing thedisease or who was with a disease, is not with the disease, but is atrisk of regression of the disease. In certain embodiments, the subjectis at a higher risk of developing the disease or at a higher risk ofregression of the disease than an average healthy member of a populationof subjects.

The terms “condition,” “disease,” and “disorder” are usedinterchangeably.

In general, the “effective amount” of an active ingredient refers to anamount sufficient to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof a polymer of the invention may vary depending on such factors as thedesired biological endpoint, the pharmacokinetics of the activeingredient, the disease being treated, the mode of administration, andthe age, health, and condition of the subject. An effective amountencompasses therapeutic and prophylactic treatment.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of an active ingredient is an amount sufficient toprovide a therapeutic benefit in the treatment of a disease, disorder orcondition, or to delay or minimize one or more symptoms associated withthe disease, disorder or condition. A therapeutically effective amountof an active ingredient means an amount of the active ingredient, aloneor in combination with other agents or therapies, which provides atherapeutic benefit in the treatment of the disease, disorder orcondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of disease or condition, or enhances the therapeutic efficacy ofanother therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of an active ingredient is an amount sufficient toprevent a disease, disorder or condition, or one or more symptomsassociated with the disease, disorder or condition, or prevent itsrecurrence. A prophylactically effective amount of an active ingredientmeans an amount of the active ingredient, alone or in combination withother agents or therapies, which provides a prophylactic benefit in theprevention of the disease, disorder or condition. The term“prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

EXAMPLES Example 1

The chemical composition of linear PBAEs was optimized for mRNA deliveryto A549 lung epithelial cells using a sub-set of monomers shown in FIG.1A. Linear PBAEs were synthesized via Michael addition of a diacrylate(A2) to a primary or bis-secondary amine (B2) (FIG. 1B). The polymerswere then end-capped with amines. It was established that PBAE‘DD90-118’ displayed the highest transfection efficiency in vitro (FIG.1C). DD90-118 was selected to further investigate the effect ofmanipulating polymer architecture.

Example 2

Hyperbranched PBAE polymers were synthesized using an A₂, B₂, BB′₂synthesis strategy. Monombers DD, 90, and a multifunctional amine,N-methyl 1,3 diaminopropane (BB′₂) were reacted (FIG. 2A). To controlDB, the monomer feed ratio of amine ‘90’ (B₂) to branching amine (BB′₂)was varied to generate hyperbranched PBAEs with a DB of 0.1, 0.2 and 0.3(FIG. 2B) as confirmed by ¹H NMR which showed good agreement betweentheoretical and experimental values. Hyperbranched (hDD90) and linearDD90 polymers were end capped with di-primary amine 118. The linearDD90-118 and three hDD90-118 polymers had molecular weights between15-24 kDa as determined by triple detection GPC.

¹H NMR analysis indicated that an increase in DB correlates with anincrease in terminal ‘end-cap’ amine groups (FIG. 6A). These end-capamines consequently increase the density of primary and secondary aminesin the PBAE and may influence polymer efficacy as a transfection reagentat various stages. Initially, during nanoparticle formulation, thecationic polymer protects nucleic acid cargo through electrostaticcondensation to prevent degradation by nucleases. This was illustratedin a gel retardation assay, where the hyperbranched PBAEs were able tokeep mRNA protected within polyplex and prevent movement through the gelat a lower N/P ratio of 5, whereas the linear polymer could not. (FIGS.7A and 7B).

On the basis of the high transfection efficiency observed in vitro (FIG.2C) and effective mRNA binding (FIGS. 7A and 7B), hDD90-118 with a DB of0.3 (referred to as “the hPBAE”) was selected to take forward for invivo nebulization of mRNA. Properties pertinent to aerosol formulationwere characterized, including polymer aqueous solubility andnanoparticle stability. Using UV spectroscopy, an increase in aqueoussolubility for the hPBAE (30.8 mg/mL) was observed compared as to linearDD90-118 (25.4 mg/mL) at pH 5.2, notably in the absence of solventstraditionally required to increase solubility such as dimethyl sulfoxide(FIG. 8 ). This increase in solubility of the hPBAE is likely due to theincrease in polar end groups (FIGS. 6A and 6B) and is a substantialbenefit, particularly for nebulized delivery where concentrated dosesare required for clinically relevant levels of gene delivery. Particlestability against pH was tested, and it was found that particle diameterremained below 100 nm between pH 4 to 7 for both branched and linearPBAEs. However, upon increasing pH, hPBAE nanoparticles remained below120 nm up to pH 8.5 compared to linear PBAEs, which began to grow insize at pH 7.5 (168 nm) and aggregated above pH 8 (FIG. 3 ). The loss ofparticle stability correlates to a reduction in zeta potential below +30mV, which occurs at around pH 7.5 for linear PBAE and pH 8.5 forbranched (FIG. 3 ) suggesting that a charge above +30 mV is required forcolloidal stability of these nanoparticles in the absence of stericstabilization such as that afforded by polyethylene glycol (PEG)coatings.

Example 3

Polyplexes containing firefly luciferase-encoding mRNA were preparedwith linear or branched DD90-118 at a polymer concentration of 25 mg/mL,and used a 50:1 mass ratio of polymer to mRNA. bPEI 25 kDa was alsoprepared at an equivalent N/P ratio of 57. A vibrating mesh nebulizerwas used to aerosolize the nanoparticle formulations and afterconfirming that particle size and charge remained stable after thisprocess (FIGS. 9A and 9B), 1 mL of the formulation was nebulized to micevia a whole-body chamber (FIG. 4A). Both linear and branched PBAE werefound to transfect the lung and express significantly higherbioluminescence than the bPEI 25 kDa. In addition, the nanoparticleswere lyophilized with sucrose as a cryoprotectant, stored at −80° C. andreconstituted in water to a concentration of 0.5 mg/mL mRNA prior tonebulization, adding a significant benefit for ease of preparation.Particles were used 48 h post-lyophilization. However, there ispotential for longer term storage as hPBAE particles remained stable andeffective in vivo after 14 and 90 days of storage at −80° C. (FIGS. 9Aand 9B). After 24 hours, organs were harvested from mice andbioluminescence was found to be significantly higher in the lung whenluciferase mRNA was delivered using hPBAE polyplexes (3.2×10⁵p/s/cm²/sr) compared to bPEI (2.4×10⁴, p=0.003) (FIG. 4B). Uniformdistribution of bioluminescence was achieved throughout all 5 lobes ofthe lung (FIG. 4C) and translation of luciferase mRNA was localized tothe lung with no bioluminescence observed in the liver, kidneys, spleenor heart (FIG. 4D). All nebulized delivery vectors were well toleratedin mice as indicated by no significant weight loss (FIG. 4E) and absenceof lung inflammation assessed by histology at 48 h post-nebulization(FIG. 4F).

A sensitive cell specific approach using Ai14 reporter mice was used toidentify the lung cell subtype that was being transfected by thebranched hDD90-118 formulation. The mice harbor a loxP-flanked stopcassette that controls gene expression of the highly fluorescenttdTomato protein and is only expressed in the presence of Crerecombinase. hDD90-118 (DB 0.3) polyplexes containing mRNA encoding forCre-recombinase were nebulized to mice. After 7 days, lungs wereanalyzed by flow cytometry, revealing that the lung epithelial cellpopulation expressed the majority of tdTomato (FIG. 4G), compared toendothelial or immune cells (FIGS. 10A, 10B and 10C).

To determine if the lyophilized formulations could also be utilized inan alternative, commonly-reported delivery route, luciferase mRNA wasadministered at a dose of 0.5 mg/kg to mice via intravenous injection.Interestingly, after systemic delivery, bioluminescence remained highestin the lung for the cationic polyplexes (FIG. 5 ). Bioluminescence inthe lung was significantly higher in mice administered with hDD90-118polyplexes (4.0×10⁶) compared to bPEI (1.1×10⁶, p=0.01) and caused lowerweight loss than the linear DD90-118, indicating reduced toxicity (FIG.5C). Further analysis of the lung by flow cytometry found that hDD90-118polyplexes transfected 51.8% of the lung endothelial cell population and13.8% of the epithelial cell population (FIGS. 10A, 10B and 10C). Incontrast to local nebulized lung delivery, systemic administration alsogenerated detectable levels of bioluminescence in the spleen (FIG. 5A).

Example 4: Poly(Beta Amino Ester) Synthesis

Diacrylate and amine monomers were purchased from Sigma-Aldrich, AlfaAesar, TCI America and Monomer-Polymer & Dajac Labs. Linear PBAEs weresynthesized at a ratio of 1:0.95 acrylate:backbone amine. To synthesizehyperbranched PBAEs (hPBAEs), monomers were reacted at varyingstoichiometry (shown below) to achieve different degrees of branching:Molar ratio of Acrylate (e.g., Formula (A)): backbone amine (e.g.,combined Formula (B) and/or (E)): branching amine (e.g., Formula (C)):

-   -   a) 1:0.5:0.2 (DB=0.3);    -   b) 1:0.67:0.13 (DB=0.2); or    -   c) 1:0.8:0.08 (DB=0.1).

Monomers were combined in anhydrous dimethylformamide at a concentrationof 150 mg/mL and held at a temp of 40 C for 6 h followed by an increasein temp to 90 C, stirred at 90° C. for 48 hours and then allowed to coolto 30° C. The end cap amine (e.g., Formula (D)) was then added at anexcess of 1.25 molar equivalents relative to the acrylate and stirredfor a further 24 hours. The polymer was protonated and purified bydropwise precipitation into cold anhydrous diethyl ether spiked withglacial acetic acid. This was vortexed rigorously and centrifuged at1250 G for 2 mins to pellet the polymer. The supernatant was discardedand polymer washed twice more in fresh diethyl ether and dried undervacuum for 48 hours. The polymers were stored at −20° C.

Example 5: In Vitro Transfection

A549 lung epithelial cells (ATCC) were seeded into white tissue culturepolystyrene 96 well plates (Corning) at 10,000 cells per well in 100 uLof DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum and1% antibiotic-antimycotic (Gibco). After 24 hours of incubation, eachwell was transfected with 50 ng of mRNA encoding for firefly luciferase(Shire) either naked or complexed with transfection reagents.Lipofectamine 2000 (Invitrogen) and JetPEI (Polyplus) were preparedaccording to manufacturer's instructions. Branched PEI 25 kDa (Sigma)was diluted in water to a concentration of 7.5 mg/mL and titrated to pH5. The polymer was combined with mRNA at an N/P ratio of 57, pipettedseveral times to mix and allowed to stand at room temp. for 10 minutesto allow complex formation. Protonated PBAEs were dissolved in 25 mMsodium acetate buffer (pH 5.2, Sigma) to a concentration of 10 mg/mL andcomplexed with mRNA at a mass ratio of 50 to 1 and incubated at roomtemp. for 10 minutes to allow complex formation. Each transfectionreagent was made to an mRNA concentration of 50 ng/15 uL and added towells containing 150 uL of fresh medium. Medium was changed after 4hours incubation. Cells were incubated for a further 20 hours at 37° C.,5% CO₂. Cell viability was assessed using PrestoBlue (Invitrogen)followed by luciferase expression analysis using BrightGlo (Promega)according to manufacturer's instructions.

Example 6: Lyophilization

Nanoparticles were prepared for nebulization at a mRNA concentration of0.125 mg/mL and at 0.0125 mg/mL for intravenous delivery. Sucrose(Sigma) was added to nanoparticles at 30 mg/mL, flash frozen in liquidnitrogen and lyophilized for 24 hours. Lyophilized samples were storedat −80° C. and reconstituted in water (molecular biology grade, Sigma)to a concentration of 0.5 mg/mL mRNA for nebulization and to 0.05 mg/mLfor intravenous injection.

Example 7: Animal Studies

C57BL/6 mice, 6-8 weeks old females (Charles River) were cared for inthe USDA-inspected MIT Animal Facility under federal, state, local andNIH guidelines for animal care. Nebulized delivery: An AeroNeb vibratingmesh nebulizer (Aerogen Inc.) was connected to a whole-body nebulizationchamber via a spacer half filled with silica (1-3 mm, Sigma). 3 micewere placed in the chamber. The nebulizer was loaded with 1 mL of ananoparticle formulation containing 0.5 mg/mL of IVT-mRNA encoding forfirefly luciferase (kindly provided by Shire Pharmaceuticals, LexingtonMass.). An oxygen flow rate of 20 was used to direct the aerosol alongthe spacer into the chamber until no more aerosol could be observed.Intravenous delivery: Mice were injected intravenously through the tailvein with a dose of 0.5 mg/kg mRNA encoding for firefly luciferase.After 24 hours, mice were injected with 0.2 mg/g of Luciferin(Xenolight, PerkinElmer) intra-peritoneally and sacrificed 10 minutespost-injection. Organs were harvested and luciferase expression assessedby bioluminescence imaging (Xenogen IVIS Spectrum Imager).

Example 8: Flow Cytometry Studies with Ai14 Cre Reporter Mice

B6. Cg-Gt(ROSA)26Sor^(tm14(CAG-tdTomato)Hze)/J mice (Jackson Laboratory,Bar Harbor, Me.) were nebulized with 1 mL of hPBAE nanoparticles loadedwith 0.5 mg/mL mRNA encoding for Cre Recombinase (Trilink, NLS-Cre, 5meC, Ψ). Nanoparticles were prepared according to the protocol describedearlier. As a control, genetically normal C57BL/6 mice were nebulizedwith water containing 100 mM sodium acetate and 60 mg/mL glucose. Micewere sacrificed 7 days post-nebulization and the lungs harvested. Thelungs were minced and incubated for 1 hr at 37° C. in PBS buffer (Gibco)containing 0.92 M HEPES (Gibco), 201.3 units/mL collagenase I (Sigma),566.1 units/mL collagenase XI (Sigma), and 50.3 units/mL DNase I(Sigma). The digested tissue was filtered through a 70 μM nylon cellstrainer and treated with red blood cell (RBC) lysis buffer for 5minutes. Following RBC lysis, the cell suspension was centrifuged at 400G and the pellet re-suspended in PBS containing 0.5% bovine serumalbumin and filtered through a 40 μM cell strainer. The single cellsuspension was centrifuged again, pellet re-suspended, and thenincubated for 30 minutes at 4° C. with antibodies against epithelial(EPCAM-APC), endothelial (CD31-AF488), and immune (CD45-BV421) cellmarkers at a 1:300 dilution (all antibodies obtained from BioLegend, SanDiego, Calif.). The cells were then analyzed using an LSR HTS-II flowcytometer (BD Biosciences).

Methods

Proton nuclear magnetic resonance (H¹ NMR): Polymer was dissolved indeuterated dimethyl sulfoxide (DMSO-d6, Sigma). Spectra were acquiredusing a Bruker spectrometer (400 MHz). Chemical shifts are reported inppm (δ) referenced against the DMSO solvent peak at 2.50 ppm. Data wereprocessed using MestReNova software (MestRelab Research).

Gel Permeation Chromatography: Non-protonated PBAE polymer was dissolvedinto anhydrous tetrahydrofuran (THF) at a concentration of 5 mg/mL andfiltered through a 0.2 pm PTFE filter. Absolute molecular weight andintrinsic viscosity was recorded using an Agilent 1260 Infinitymulti-detector instrument equipped with a viscometer and detectors forrefractive index and light scattering. Data was acquired at 35° C. at aflow rate of 1 mL/min. Analysis was performed using OmniSEC software.

Gel electrophoresis retardation assay: Polyplexes were prepared at amRNA concentration of 0.1 mg/mL and at polymer mass ratios to mRNA of2.5, 5.0 and 7.5 to 1 in water (Molecular biology grade, Sigma). A 20 μLsample was loaded per well of a SybrGold 1% agarose EX E-Gel and run onan iBase (Invitrogen) for 10 minutes, then imaged on a BioRad ChemiDocsystem.

Solubility Assay: A calibration curve was first generated by dissolvingPBAE polymer in 25 mM sodium acetate (NaOAc) buffer (Sigma, pH 5.2) at 6mg/mL and 3 mg/mL. These were serially diluted 1 in 2 until aconcentration of 0.75 mg/mL was obtained. Absorbance at 250 nm of 100 μLof each dilution was measured in a UV transparent 96 well polystyrolplate (Corning). To test solubility, a supersaturated sample wasprepared by adding excess polymer (40 mg/mL) into 25 mM NaOAc buffer andsonicated for 30 minutes. The sample was filtered through a 0.2 pm PTFEfilter and serial dilutions of the filtrate prepared. Absorbance wasmeasured at 250 nm and concentration of the samples calculated using thecalibration curve.

Particle Stability over varying pH: Polyplexes were prepared at an mRNAconcentration of 0.25 mg/mL and PBAE to mRNA mass ratio of 50 to 1 in 25mM NaOAc, 0.8 mL of particles were added to 9.2 mL of deionized water.Zeta potential and particle diameter was measured using a MalvernZetasizer Nano instrument equipped with a pH auto-titrator and probe(MPT-2). Hydrochloric acid and sodium hydroxide were used to titrate pHfrom 4 to 11.

Histology: After administering 3 repeat doses to mice, they weresacrificed at 24 hours post-nebulization. Lungs were inflated with 4%paraformaldehyde injected through the trachea. The trachea was tied andlungs submerged in 4% paraformaldehyde overnight. Lungs were stored in70% ethanol until being embedded in a paraffin block. Sections ofembedded tissue were then mounted on slides and stained with hematoxylinand eosin. Slides were viewed using a Zeiss Axioplan II uprightmicroscope at 20× magnification.

Flow Cytometry Studies with Cy5-labeled mRNA. C57 BL/6 mice wereinjected intravenously with polyplexes loaded with Cyanine 5-labeledluciferase-encoding mRNA (Trilink, 5 meC, ψ) at a dose of 0.5 mg/kgaccording to a previous protocol.³ Control mice were injected withphosphate buffered saline. Mice were sacrificed 1 hour after injectionand perfused with PBS. The lungs were harvested and imaged on an IVISimaging apparatus. Following imaging, lungs were processed into a singlecell suspension and analyzed as described in the Ai14 reporter mousemethods section. Antibodies used were CD45-BV510 (immune cells),CD31-Pacific Blue (endothelial cells), and EpCAM-PE (epithelial cells).Cell populations were identified as follows: Immune (CD45+), Endothelial(CD45−, CD31+, EpCAM−), and Epithelial (CD45−, CD31−, EpCAM+).

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps.

Where ranges are given, endpoints are included. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

The invention claimed is:
 1. A composition comprising a poly(beta-aminoester) polymer, or a salt thereof, the polymer comprising: a diradicalof Formula (A) having the structure:

a diradical of Formula (B) having the structure:

a triradical of Formula (C) having the structure:

and a radical of Formula (D) having the structure:

wherein: each diradical of Formula (A) has two points of attachment toradicals independently selected from Formulae (B), (C), and (D); eachdiradical of Formula (B) has two points of attachment to a diradical ofFormula (A); each triradical of Formula (C) has three points ofattachment to a diradical of Formula (A); and each radical of Formula(D) has one point of attachment to a diradical of Formula (A).
 2. Thecomposition of claim 1, wherein the polymer comprises radicals ofFormulae (A), (B), (C), and (D) in a ratio of about 1:0.5:0.2:0.39;1:0.67:0.13:0.27; 1:0.8:0.08:0.16; or 1:0.94:0:0.07.
 3. The compositionof claim 1, wherein the polymer has a molecular weight in the range of10-40 kDa.
 4. The composition of claim 1, wherein the polymer has adegree of branching (DB) in the range of 0.1-0.7.
 5. The composition ofclaim 1, further comprising an agent.
 6. The composition of claim 5,wherein the agent is a polynucleotide.
 7. The composition of claim 6,wherein the polynucleotide is DNA.
 8. The composition of claim 6,wherein the polynucleotide is RNA.
 9. The composition of claim 8,wherein the RNA is mRNA, siRNA, ssRNA, dsRNA, shRNA, or miRNA.
 10. Thecomposition of claim 9, wherein the RNA is siRNA.
 11. The composition ofclaim 6, wherein the polynucleotide encodes an antigenic peptide orprotein.
 12. The composition of claim 11, wherein the antigenic peptideor protein is derived from a virus.
 13. The composition of claim 1, inthe form of a particle.
 14. The composition of claim 13, wherein theparticle is a microparticle, nanoparticle, micelle, liposome, orlipoplex.
 15. The composition of claim 1, formulated for nebulization,aerosol delivery, or intranasal delivery.
 16. A nasal spray comprisingthe composition of claim
 5. 17. A method of delivering a polynucleotideto a cell, comprising contacting the cell with the composition of claim6.
 18. The method of claim 17, wherein the cell is a lung epithelialcell.
 19. A method of treating a disease or disorder in a subject inneed of such treatment, comprising administering to the subject thecomposition of claim
 5. 20. The method of claim 19, wherein the diseaseor disorder is of the lungs.
 21. The method of claim 19, wherein thedisease or disorder is lung cancer asthma, chronic obstructive pulmonarydisease, or pulmonary fibrosis.
 22. The method of claim 19, wherein saidadministration is intranasal administration.
 23. The composition ofclaim 9, wherein the RNA is mRNA.